Human Artificial Chromosome Vector

ABSTRACT

A human artificial chromosome vector comprising a gene encoding a human antibody heavy chain, a gene encoding a human antibody light chain, and a gene encoding an IgM heavy chain constant region derived from a nonhuman animal.

TECHNICAL FIELD

The present invention relates to a human artificial chromosome vectorcomprising a human antibody heavy chain gene, a human antibody lightchain gene, and an IgM heavy chain constant region gene derived from anon-human animal, an animal having the human artificial chromosomevector, and a method for producing a human antibody.

BACKGROUND ART

The development of a technology of producing a chimeric animal by fusingmicronuclei comprising human chromosome fragments with cells havingpluripotent differentiation to obtain hybrid cells allows to prepare anon-human animal maintaining large exogenous genes to be produced(Non-Patent Document 1 and Patent Document 1).

Subsequently, a method for constructing a desired human artificialchromosome (hereinafter abbreviated to HAC) vector to produce thenon-human animal has been proposed, by which the HAC including a widerange of human antibody gene loci has been established.

First, as a method for modifying a chromosome fragment to be introducedinto a non-human animal, a technology of preparing a deleted chromosomewith a high efficiency by inserting a telomere sequence into a desiredsequence on a human chromosome kept in a chicken DT40 cell by genetargeting has been developed (Patent Document 2)

Further, in a process of maintaining a mouse cell keeping a humanchromosome, it has been found that a fragment SC20 comprising anantibody heavy chain gene locus derived from the human chromosome 14 maybe obtained, and the fragment is stably maintained in an embryonic stem(ES) cell and an individual of the mouse and has high transmissionefficiency to progeny (Patent Document 2).

Therefore, a λHAC comprising a human antibody heavy chain and a humanantibody λ chain was constructed by translocating a fragment comprisingthe antibody type light chain gene locus on the human chromosome 22through the Cre/loxP site specific recombination system using such SC20as a basic skeleton of a vector (Non-Patent Document 2 and PatentDocument 3).

The λHAC has the stability and transmission efficiency to progeny almostequivalent to the SC20 and a chimeric mouse which stably maintains aλHAC is produced by introducing the λHAC into the mouse ES cells(Non-Patent Document 2 and Patent Document 3). It is now possible toconstruct a HAC vector including a human chromosome region having aspecific megabase (Mb) size by the method.

Further, for the purpose of removing chromosome regions which adverselyaffect the generation of a chromosome-introduced animal, chromosomefragments ΔHAC and ΔΔHAC with an optimal size including the antibody λtype light chain (λ chain) gene region were prepared (Patent Document4), based on the structural information of the human chromosome 22(Non-Patent Document 3).

It was confirmed that the ΔHAC and ΔΔHAC include regions having 2.5 Mband 1.5 Mb sizes, respectively, which are shorter than the periphery ofthe antibody λ type light chain gene region on the MAC, 10 Mb, to imparta transmission efficiency to progeny higher than that of the λHAC(Patent Document 4).

Meanwhile, human polyclonal antibodies currently used for treatment andprevention of various diseases are prepared from a serum pool obtainedfrom a plurality of human donors. For this reason, the performance ofthe human polyclonal antibodies depends largely on human donor sera as asupplying source, and thus a process of selecting a donor having adesired antigen reactivity or titer is required in preparing.

In addition, due to factors such as the kind of antigen, the number ofexposure to an immunogen, and the amount of donor serum which may becollected, in preparing a polyclonal antibody, the development of theuse thereof is limited. Therefore, a transgenic animal having a humanantibody gene locus as a means for producing human polyclonal antibodieshas been prepared.

Due to its body size, an ungulate animal is useful as a source ofsupplying a large quantity of human polyclonal antibodies. So far, inorder to produce human polyclonal antibodies, a bovine into which theabove-described HAC, specifically ΔHAC and ΔΔHAC are introduced toproduce human polyclonal antibodies, is known (Non-Patent Document 4 andPatent Document 5).

In the neonatal sera of the bovine into which these HACs wereintroduced, 13 to 258 ng/mL of the human immunoglobulin (Ig) G wasproduced (Non-Patent Document 4).

Subsequently, in order to eliminate bovine antibodies produced in abovine living body, gene targeting was carried out on IGHM and IGHML1encoding the functional IgM genes among bovine endogenous antibody heavychain genes and as a result, a bovine in which antibody heavy chainswere knockout was known (Non-Patent Documents 5 and 6 and PatentDocuments 6 and 7).

When ΔΔHAC was introduced into the obtained antibody heavy chaindouble-knockout bovine (IgHM^(−/−)/IgHML1^(−/−) bovine), it was found7.1 μg/mL of a human IgG was produced in a serum of a 14-day-old calf(Patent Document 7).

In addition, a bovine into which κHAC which is a HAC vector having ahuman antibody heavy chain gene locus and a human antibody κ type lightchain (κ chain) gene locus was introduced was produced (Non-PatentDocument 6 and Patent Document 8). κHAC is a vector constructed bytranslocating a fragment comprising the antibody κ chain gene locus onthe human chromosome 2 onto the SC20. In FIG. 1, a schematic view ofκHAC is shown.

Among IgHM^(−/−)/IgHML^(−/−) bovines into which κHAC was introduced,clone 468 which was the highest antibody producing individual constantlyproduced a human IgG at 1 g/L or more in the serum from the 84 daysafter birth, and exhibited a titer exceeding 2 g/L at the 210 days afterbirth.

However, a technology of stably producing an individual which exhibitshigh titer as described above has not been known, and thus there is aneed for animals which produce human antibodies with higher efficiencyor a technology which can stably produce these animals.

CITATION LIST Patent Document

-   Patent Document 1: WO97/07671-   Patent Document 2: WO98/37757-   Patent Document 3: WO00/10383-   Patent Document 4: WO02/92812-   Patent Document 5: WO2002/70648-   Patent Document 6: WO03/97812-   Patent Document 7: WO05/104835-   Patent Document 8: WO09/111086

Non-Patent Document

-   Non-Patent Document 1: Tomizuka et al., Nature Genetics, 16,    133-143, 1997-   Non-Patent Document 2: Kuroiwa et al., Nature Biotechnology, 18,    1086-1090, 2000-   Non-Patent Document 3: Dunham et al., Nature, 402, 489-495, 1999-   Non-Patent Document 4: Kuroiwa et al., Nature Biotechnology, 20,    889-894, 2002-   Non-Patent Document 5: Kuroiwa et al., Nature Genetics, 36, 775-780,    2004-   Non-Patent Document 6: Kuroiwa et al., Nature Biotechnology, 27,    173-181, 2009

Summary of Invention Technical Problem

It is an object of the present invention to provide an animal forproducing human antibodies efficiently, a method for stably supplyingthe animal, and a method for producing human antibodies with highefficiency.

Solution to Problems

Considering the above object, the present inventors modified the HACvector for the purpose of achieving high production amount of the humanIgG and stably producing a bovine individual with a high titer, comparedto the conventional HAC.

That is, the present invention relates to the following (1) to (8).

(1) A human artificial chromosome vector comprising a gene encoding ahuman antibody heavy chain, a gene encoding a human antibody lightchain, and a gene encoding an IgM heavy chain constant region derivedfrom a non-human animal.(2) The human artificial chromosome vector described in (1), comprisinga gene encoding a human antibody surrogate light chain(3) The human artificial chromosome vector described in (2), in which agene encoding a human antibody surrogate light chain is the VpreB geneand the λ5 gene.(4) The human artificial chromosome vector described in any one of (1)to (3), in which the gene encoding the non-human animal-derived IgMheavy chain constant region is the bovine-derived IGHM.(5) An animal having the human artificial chromosome vector described inany one of (1) to (3).(6) A bovine having the human artificial chromosome vector described in(4).(7) A method for producing a human antibody, comprising: administering atarget antigen into an animal described in (5) to produce and accumulatethe human antibody specific to the antigen in serum of the animal, andrecovering the human antibody specific to the antigen from the serum.(8) A method for producing a human antibody, comprising: administering atarget antigen into a bovine described in (6) to produce and accumulatethe human antibody specific to the antigen in serum of the bovine, andrecovering the human antibody specific to the antigen from the serum.

Advantageous Effects of the Invention

Human antibodies may be produced with high efficiency by introducing thehuman artificial chromosome vector comprising a gene encoding the humanantibody heavy chain, a gene encoding the human antibody light chain,and a gene encoding the IgM heavy chain constant region derived from anon-human animal of the present invention into an animal as compared tothe vector produced by the conventional HAC technology. In addition, ananimal which is capable of producing a human antibody with highefficiency may be stably produced by introducing the human artificialchromosome vector of the present invention into an animal.

Furthermore, as a preferred embodiment, when a human artificialchromosome vector which further comprises a human antibody surrogatelight chain gene in addition to a gene encoding the human antibody heavychain, a gene encoding the human antibody light chain, and a geneencoding the non-human animal-derived IgM heavy chain constant region,is introduced into an animal, a human antibody may be produced with afurther higher efficiency, and an animal which is capable of producing ahuman antibody with such high efficiency may be stably produced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) illustrates a schematic view of κHAC. (B) illustrates aschematic view of an IgM to be formed on a B cell membrane when κHAC isintroduced into an animal. In (B), the dotted-line represents ahuman-derived IgM part, and the solid-line represents a bovine-derivedIgM part.

FIG. 2 (A) illustrates a schematic view of KcHAC. (B) illustrates aschematic view of an IgM to be formed on the B cell membrane when KcHACis introduced into an animal. In (B), the dotted-line represents ahuman-derived IgM part, and the solid-line represents a bovine-derivedIgM part.

FIG. 3 (A) illustrates a schematic view of cKSL-HACΔ. (B) illustrates aschematic view of an IgM to be formed on the B cell membrane whencKSL-HACΔ is introduced into an animal. In (B), the dotted-linerepresents a human-derived IgM part, and the solid-line represents abovine-derived IgM part.

FIG. 4 illustrates a schematic view of a targeting vectorpTEL′hisDpurolox²²⁷²F9R9.

FIG. 5 illustrates a schematic view of a targeting vectorpTELCAGzeoSLF2R2.

FIG. 6 illustrates a schematic view of a targeting vectorp553CAG^(lox2272)BsrDT.

FIG. 7 illustrates a schematic view of a targeting vectorpSC355CAG^(lox511)hisDDT.

FIG. 8 illustrates a schematic view of a targeting vectorp14CEN(FR)hygpuro^(lox511)DT

FIG. 9 illustrates a schematic view of a targeting vectorpRNR2^(l0xP)bsrDT.

FIG. 10 illustrates a schematic view of a targeting vector pCH1CAGzeoDT.

FIG. 11 illustrates a schematic view of a targeting vector pCH2CAGzeoDT.

FIG. 12 illustrates an outline of a method for modifying the humanchromosome 2 in a chicken DT40 cell.

FIG. 13 illustrates an outline of a method for modifying the humanchromosome 22 in a chicken DT40 cell.

FIG. 14 illustrates an outline of a method for constructing the SLKHfragment in a DT40 hybrid cell.

FIG. 15 illustrates an outline of a method for constructing the CH2Dfragment in a DT40 cell.

FIG. 16 illustrates an outline of a method for modifying the humanchromosome 14 in a DT40 cell.

FIG. 17 illustrates an outline of a method for constructing cKSL-HACΔ ina DT40 hybrid cell.

FIG. 18 illustrates an outline of a method for constructing cHAC infibroblast cells.

FIG. 19 illustrates a total amount of the human IgG in each serum of6-month-old HAC bovines.

FIG. 20 illustrates the result of a CEM cell specific ELISA assay.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention relates to (1) a human artificial chromosomevector comprising a human antibody heavy chain gene, a human antibodylight chain gene, and, a non-human animal-derived 1 gM heavy chainconstant region gene, (2) an animal having the human artificialchromosome vector, and (3) a method for producing a human antibody,comprising administering a target antigen into the animal to produce andaccumulate the antigen-specific human antibody in the serum of theanimal, and recovering the antigen-specific human antibody from theserum.

1. The Human Artificial Chromosome Vector of the Present Invention

In the present invention, “the human artificial chromosome vector”refers to a vector which comprises a human chromosome-derived centromeresequence, a telomere sequence, and a replication origin and existsindependently from a chromosome of a host cell in a nucleus of the hostcell.

Specifically, the vector refers to a human artificial chromosome (HAC)vector prepared by translocating a desired region on the humanchromosome into a stable human chromosome fragment.

Specific examples of a method for preparing a HAC include a methodcomprising: inserting a telomere sequence and a sequence loxP recognizedby a Cre recombinant enzyme such that a desired region on a humanchromosome or a chromosome fragment may be included, and binding aregion inserted between the telomere sequence of the chromosome or thechromosome fragment and the loxP sequence to a region inserted between atelomere sequence on another chromosome or chromosome fragment(preferably a chromosome fragment which is stable in the nucleus and hasa high transmission efficiency to progeny) and the loxP sequence bytranslocation (Kuroiwa et al., Nature Biotechnology, 18, 1086-1090, 2000and WO No. 00/10383).

(Human Antibody Heavy Chain Gene)

The human artificial chromosome vector of the present inventioncomprises a human antibody heavy chain gene. In the present invention,“the human antibody heavy chain gene” refers to a gene which, among twoidentical heavy chains and two identical light chains constituting ahuman immunoglobulin molecule, encodes the former.

Specific examples of the human antibody heavy chain gene include a geneencoding a variable region of the heavy chain and genes encoding the ychain, β-1 chain, α chain, δ chain, and ε chain which determine thestructure of the constant region.

In the artificial chromosome vector of the present invention,“comprising the human antibody heavy chain gene” refers to comprising aDNA encoding the human antibody heavy chain gene. The human artificialchromosome vector of the present invention may be prepared by insertinga DNA encoding the human antibody heavy chain gene into any position orinserting or ligating a chromosome fragment comprising a DNA encodingthe human antibody heavy chain gene.

The variable region gene and the constant region gene of the humanantibody heavy chain form a cluster and is positioned at 14q32 on thehuman chromosome 14. Therefore, the artificial chromosome vector of thepresent invention preferably comprises a human chromosome 14 fragment,more preferably a human chromosome 14 fragment at which the variableregion gene and the constant region gene of the human antibody heavychain are positioned, and further preferably a human chromosome 14fragment comprising the 14q32 region.

(Human Antibody Light Chain Gene)

The human artificial chromosome vector of the present inventioncomprises a human antibody light chain gene. In the present invention,“the human antibody light chain gene” refers to a gene that, among twoidentical heavy chains and two identical light chains constituting ahuman immunoglobulin molecule, encodes the latter.

Specific examples of the human antibody light chain gene include twotypes of genes, i.e., the κ chain gene and the λ chain gene. The gene ofeach chain comprises a gene encoding a variable region and a geneencoding a constant region.

The human artificial chromosome vector of the present invention maycomprise either κ chain gene or the λ chain gene only, or both of thegenes as the human antibody light chain gene.

In the human artificial chromosome vector of the present invention,“comprising the human antibody light chain gene” refers to comprising aDNA encoding the human antibody light chain gene. The human artificialchromosome vector of the present invention may be prepared by insertinga DNA encoding the human antibody light chain gene into any position orinserting or ligating a chromosome fragment comprising a DNA encodingthe human antibody light chain gene.

The variable region gene and constant region of the κ chain and the λchain forms a cluster to be positioned on a chromosome. The κ chain genecluster is positioned at 2p11.2 of the human chromosome 2 (Gottfrie etal., Genomics, 16. 512-514, 1993) and the λ chain gene cluster ispositioned at 22q11.2-12 of the human chromosome 22 (Collins et al.,Nature, 377, 367-379, 1995).

Therefore, the artificial chromosome vector of the present inventionpreferably comprises a human chromosome 2 fragment, more preferably ahuman chromosome 2 fragment at which the κ chain gene cluster ispositioned, and even more preferably a human chromosome 2 fragmentcomprising the 2p11.2-12 region.

In addition, the artificial chromosome vector of the present inventionpreferably comprises a human chromosome 22 fragment, more preferably ahuman chromosome 22 fragment at which the chain gene cluster ispositioned, and even more preferably a human chromosome 22 fragmentcomprising the 22q11.2 region.

(IgM Heavy Chain Constant Region Gene)

The human artificial chromosome vector of the present inventioncomprises a non-human animal-derived IgM heavy chain constant regiongene. The non-human animal is not particularly limited so long as theanimal is a non-human animal which can be a host into which the humanartificial chromosome vector of the present invention is introduced, andmay be any of ungulates such as cows, horses, goats, sheep, and pigs;rodents such as mice, rats, and rabbits; poultry such as chickens,domestic ducks, and geese.

The non-human animal is preferably a non-human mammalian animal, morepreferably an ungulate animal, and furthermore preferably a bovine.

In the present invention, “the IgM heavy chain constant region gene”refers to a gene encoding the IgM heavy chain constant region. The IgMheavy chain constant region promotes the generation of the B cell byinteracting with the B cell membrane molecule Igα/Igβ to cause a signaltransduction in cells. Specific examples of the IgM heavy chain constantregion gene include genes encoding constant region domains such as CH1,CH2, CH3, and CH4, and the B-cell transmembrane domains such as TM1 andTM2.

The IgM heavy chain constant region gene derived from a non-human animalwhich is comprised in the human artificial chromosome vector of thepresent invention is not particularly limited so long as the region isin a range which may sufficiently induce the signal of the B cellgeneration in the above-described IgM heavy chain constant region, butpreferably comprises TM1 domain and TM2 domain derived from a non-humananimal, and more preferably genes encoding CH2 domain, CH3 domain, CH4domain, TM1 domain, and TM2 domain which are derived from a non-humananimal.

In the human artificial chromosome vector of the present invention,“comprising the non-human animal-derived IgM heavy chain constant regiongene” refers to comprising a DNA encoding a non-human animal-derived IgMheavy chain constant region gene.

The human artificial chromosome vector of the present invention may beprepared by inserting a DNA encoding IgM heavy chain constant regiongene derived from a non-human animal into any position, or ligating achromosome fragment comprising a DNA encoding a human IgM heavy chainconstant region gene.

In particular, it is preferred that some of the DNA encoding the humanIgM heavy chain constant region gene on the human artificial chromosomeis substituted with some of the DNA encoding the non-humananimal-derived IgM heavy chain constant region gene.

Specifically, when a part of the DNA encoding the human IgM heavy chainconstant region gene on the human artificial chromosome is substitutedwith a part of the DNA encoding IgM heavy chain constant region genederived from a non-human animal, the DNAs encoding the TMI domain andthe TM2 domain in the human IgM heavy chain constant region gene on thehuman artificial chromosome are preferably substituted with the DNAsencoding the TM1 domain and the TM2 domain in the non-human IgM heavychain constant region gene, respectively; and more preferably the DNAsencoding the CHI domain, the CH2 domain, the CH3 domain, the CH4 domain,the TM1 domain, and the TM2 domain in the human IgM heavy chain constantregion gene are substituted with the DNAs encoding the CH1 domain theCH2 domain, the CH3 domain, the CH4 domain, the TM1 domain, and the TM2domain in the non-human IgM heavy chain constant region gene,respectively; or the DNAs encoding the CH2 domain, the CH3 domain, theCH4 domain, the TM1 domain, and the TM2 domain in the human IgM heavychain constant region gene on the human artificial chromosome aresubstituted with the DNAs encoding the CH2 domain, the CH3 domain, theCH4 domain, the TM1 domain, and the TM2 domain of the non-human IgMheavy chain constant region gene, respectively.

The IgM heavy chain constant region gene derived from a non-human animalis preferably the IgM heavy chain constant region gene of a non-humanmammalian animal, more preferably the IgM heavy chain constant regiongene of an ungulate animal, and furthermore preferably the IgM heavychain constant region gene of a bovine.

The IgM heavy chain constant region gene of the bovine is preferably agene encoding a bovine IgM heavy chain constant region which is includedin an IGHM region at which a bovine endogenous IgM heavy chain gene ispositioned (derived from IGHM) or a gene encoding a bovine IgM heavychain constant region in an IGHML1 region (derived from IGHML1), andmore preferably a gene encoding a bovine IgM heavy chain constant regionwhich is included in the IGHM region.

(Human Antibody Surrogate Light Chain Gene)

The human artificial chromosome vector of the present inventioncomprises a human antibody surrogate light chain gene. In the presentinvention, “the human antibody surrogate light chain gene” refers to agene encoding an imaginary antibody light chain which is associated withan antibody heavy chain produced by a gene reconstitution in the humanpro-B cell to constitute the pre-B cell receptor (preBCR).

Specific examples of the human antibody surrogate light chain geneinclude the VpreB gene and the gene. The human artificial chromosomevector of the present invention preferably comprises the VpreB gene andthe λ5 gene as a human antibody surrogate light chain gene.

The VpreB gene of the present invention preferably comprises either orboth of the VpreB1 gene and the VpreB3 gene and more preferably both ofthe VpreB1 gene and the VpreB3 gene.

In the human artificial chromosome vector of the present invention,“comprising the human antibody surrogate light chain gene” refers tocomprising a DNA encoding the human antibody surrogate light chain gene.The human artificial chromosome vector of the present invention may beprepared by inserting a DNA encoding the human antibody surrogate lightchain gene into any position or inserting or ligating a chromosomefragment comprising a DNA encoding the human antibody surrogate lightchain gene.

Any of the VpreB gene and the λ5 gene is positioned within the humanantibody λ chain gene locus at 22q11.2 of the human chromosome 22.Therefore, the human artificial chromosome vector of the presentinvention preferably comprises the human chromosome 22, more preferablythe human chromosome 22 comprising the VpreB gene and the λ5 gene, andfurthermore preferably the human chromosome 22 comprising the 22q11.2region.

2. Method of Constructing a Human Artificial Chromosome Vector of thePresent Invention

The human artificial chromosome vector comprising the human antibodyheavy chain gene, the human antibody light chain gene, and the humanantibody surrogate light chain gene of the present invention may beconstructed by using the following (1) to (3) methods.

(1) Construction of a Human Artificial Chromosome Fragment Comprising aHuman Antibody Heavy Chain Gene

The human artificial chromosome fragment comprising the human antibodyheavy chain gene may be constructed by isolating the human chromosome 14from a human normal cell to obtain a chromosome fragment comprising ahuman antibody heavy chain gene from the chromosome in accordance with amethod described in WO98/037757.

Specifically, although a chromosome fragment which is incidentallygenerated during an isolation process of the human chromosome 14 or aprocess of maintaining the chromosome in a cell may be isolated by amethod described in WO00/010383, a chromosome fragment may be obtainedby irradiating an ionized radiation on the human chromosome 14 to breakthe chromosome, and a chromosome fragment may be also obtained byinserting a telomere sequence into a desired position of the humanchromosome 14 to generate a deletion at the position.

Further, the thus obtained human artificial chromosome vector may beconstructed by inserting or ligating a human chromosome fragment whichdoes not comprise the human antibody heavy chain gene into a fragmentcomprising the human chromosome 14-derived human antibody heavy chaingene by using a method by Kuroiwa et al. (Kuroiwa et al., NatureBiotechnology, 18, 1086-1090, 2000 and WO00/010383).

Examples of the human artificial chromosome fragment comprising thehuman antibody heavy chain gene include SC20 (Tomizuka et al.,Proceeding of the National Academy of Sciences, 97, 722-727, 2000 andWO98/037757), λHAC (Kuroiwa et al., Nature Biotechnology, 18, 1086-1090,2000 and WO00/010383), KHAC (Kuroiwa et al., Nature Biotechnology, 27,173-181, 2009 and WO09/111086), ΔHAC and ΔΔHAC (Kuroiwa et al., NatureBiotechnology, 18, 1086-1090, 2000 and WO00/10383), and the like.

(2) Construction of an Artificial Chromosome Fragment Comprising a HumanAntibody Light Chain Gene

The human artificial chromosome fragment comprising the human antibody κchain gene may be constructed by isolating the human chromosome 2 from ahuman normal cell to obtain a chromosome fragment comprising a humanantibody κ chain gene from the chromosome in accordance with a methoddescribed in WO98/037757.

Specifically, although a chromosome fragment which is incidentallygenerated during the isolation process of the human chromosome 2 or theprocess of maintaining the chromosome in a cell may be isolated by amethod described in WO00/010383, a chromosome fragment may be obtainedby irradiating an ionized radiation on the human chromosome 2 to breakthe chromosome, and a chromosome fragment may also be obtained byinserting a telomere sequence into a desired position of the humanchromosome 2 to generate a deletion at the position.

A human artificial chromosome fragment comprising the human antibody κchain gene may be constructed by inserting or ligating a fragmentcomprising the human antibody κ chain gene derived from the humanchromosome 2 into a human chromosome fragment which does not comprisethe human antibody K chain gene derived from the human chromosome 2 byusing a method by Kuroiwa et al. (Kuroiwa et al., Nature Biotechnology,27, 173-181, 2009 and WO09/111086).

Examples of the human artificial chromosome fragment comprising thehuman antibody κ chain gene to be thus obtained include κHAC (Kuroiwa etal., Nature Biotechnology, 27, 173-181, 2009 and WO09/111086).

The human artificial chromosome fragment comprising the human antibody λchain gene may be constructed by isolating the human chromosome 22 froma human normal cell to obtain a chromosome fragment comprising a humanantibody λ chain gene from the chromosome in accordance with a methoddescribed in WO98/037757.

Specifically, although a chromosome fragment which is incidentallygenerated during the isolation process of the human chromosome 22 or theprocess of maintaining the chromosome in a cell may be isolated by amethod described in WO00/010383, a chromosome fragment may be obtainedby irradiating an ionized radiation on the human chromosome 22 to breakthe chromosome, and a chromosome fragment may also be obtained byinserting a telomere sequence into a desired position of the humanchromosome 22 to generate a deletion at the position.

Further, a human artificial chromosome fragment comprising the humanantibody λ chain gene may be constructed by inserting or ligating afragment comprising the human antibody λ chain gene derived from thehuman chromosome 22 into a human chromosome fragment which does notcomprise the human antibody λ chain gene by using a method of Kuroiwa etal. (Kuroiwa et al., Nature Biotechnology, 18, 1086-1090, 2000 andWO00/010383.

Examples of the thus obtained human artificial chromosome fragmentcomprising the human antibody heavy chain gene include λHAC (Kuroiwa etal., Nature Biotechnology, 18, 1086-1090, 2000 and WO00/010383), ΔHACand ΔΔHAC (Kuroiwa et al., Nature Biotechnology, 20, 889-894, 2002 andWO02/092812).

(3) Construction of a Human Artificial Chromosome Fragment Comprising aNon-Human Animal-Derived IgM Heavy Chain Constant Region Gene

The non-human animal-derived IgM heavy chain constant region gene may becomprised into a human artificial chromosome vector of the presentinvention by substituting the human IgM heavy chain constant region geneon the human artificial chromosome fragment comprising the humanantibody heavy chain gene which is constructed by the method of theabove (1) with a non-human animal-derived IgM heavy chain constantregion gene.

Specifically, the non-human animal-derived IgM heavy chain constantregion gene may be constructed by substituting the DNAs encoding the CH2domain, the CH3 domain, the CH4 domain, the TM1 domain, and the TM2domain in the human IgM heavy chain constant region gene on the humanartificial chromosome vector comprising the human antibody heavy chaingene constructed by the method of the above (1) with the DNAs encodingthe CH2 domain, the CH3 domain, the CH4 domain, the TM1 domain, and theTM2 domain of the non-human IgM heavy chain constant region gene,respectively, by homologous recombination.

Therefore, the human artificial chromosome vector comprising the humanantibody heavy chain gene, the human antibody light chain gene, and thenon-human animal-derived IgM heavy chain constant region gene may beconstructed by substituting the DNAs encoding the CH2 domain, the CH3domain, the CH4 domain, the TM1 domain, and the TM2 domain in the humanIgM heavy chain constant region gene on the human artificial chromosomefragment, comprising the human antibody heavy chain gene in the humanartificial chromosome vector comprising the human antibody heavy chaingene and the human antibody light chain gene constructed by the methodsof the above (1) or (2), with the DNAs encoding the CH2 domain, the CH3domain, the CH4 domain, the TM1 domain, and the TM2 domain in thenon-human IgM heavy chain constant region gene, respectively, byhomologous recombination.

Alternately, the DNAs encoding the CHI domain, the CH2 domain, the CH3domain, the CH4 domain, the TM1 domain, and the TM2 domain in the humanIgM heavy chain constant region gene on the human artificial chromosomefragment, comprising the human antibody heavy chain gene in the humanartificial chromosome vector comprising the human antibody heavy chaingene and the human antibody light chain gene constructed by the methodsof the above (1) to (3), with the DNAs encoding the CHI domain, the CH2domain, the CH3 domain, the CH4 domain, the TMI domain, and the TM2domain in the non-human IgM heavy chain constant region gene,respectively, by homologous recombination.

Further, the human artificial chromosome vector comprising a geneencoding the human antibody heavy chain, a gene encoding the humanantibody light chain, and a gene encoding the non-human animal-derivedIgM heavy chain constant region of the present invention may beconstructed by using the methods of the above (1) to (3).

Specifically, the vector of the present invention may be constructed bysubstituting the human IgM heavy chain constant region gene on the humanchromosome 14 fragment comprising the human antibody heavy chain geneconstructed by the method of (1) with the non-human animal-derived IgMheavy chain constant region gene, and then ligating the human chromosome2 fragment comprising the human κ chain gene constructed by the methodof (2).

More specifically, a human artificial chromosome vector comprising thenon-human animal-derived IgM heavy chain constant region gene of thepresent invention may be constructed in the following manner. The DNAsencoding the CH1 domain, the CH2 domain, the CH3 domain, the CH4 domain,the TM1 domain, and the TM2 domain in the human IgM heavy chain constantregion gene on κHAC (Kuroiwa et al., Nature Biotechnology, 27, 173-181,2009 and WO09/111086) are substituted with the DNAs encoding the CH1domain, the CH2 domain, the CH3 domain, the CH4 domain, the TM1 domain,and the TM2 domain of the non-human IgM heavy chain constant regiongene, respectively, by homologous recombination.

Examples of the human artificial chromosome vector of the presentinvention to be constructed in this manner include KcHAC. In FIG. 2, aschematic view of KcHAC is shown. In FIG. 2, a schematic view of KcHACis shown. As shown in FIG. 2, since KcHAC comprises a gene encoding anon-human animal (bovine)-derived IgM heavy chain constant region genecompared to the structure of κHAC (FIG. 1), it can be produce humanantibodies with higher efficiency when it is introduced into an animal,and KcHAC may stably produce an animal which is capable of producinghuman antibodies with such a high efficiency.

(4) Construction of a Human Artificial Chromosome Fragment Comprising aHuman Antibody Surrogate Light Chain Gene

The human artificial chromosome fragment comprising the human antibodysurrogate light chain gene may be constructed by isolating the humanchromosome 22 from a human normal cell to obtain a chromosome fragmentcomprising a human antibody surrogate light chain gene from thechromosome in accordance with a method described in WO98/037757.

Specifically, although a chromosome fragment which is incidentallygenerated during the isolation process of the human chromosome 22 or theprocess of maintaining the chromosome in a cell may be isolated by amethod described in WO00/010383, a chromosome fragment may be obtainedby irradiating ionized radiation on the human chromosome 22 to break thechromosome, and a chromosome fragment may also be obtained by insertinga telomere sequence into a desired position of the human chromosome 22to generate a deletion at the position.

Further, a human artificial chromosome fragment comprising the humanantibody surrogate light chain gene may be constructed by inserting orligating a fragment comprising the human antibody surrogate light chaingene into a human chromosome fragment which does not comprise the humanantibody surrogate light chain gene by using a method of Kuroiwa et al.(Kuroiwa et al., Nature Biotechnology, 18, 1086-1090, 2000 andWO00/010383.

The human artificial chromosome vector comprising a gene encoding thehuman antibody heavy chain, a gene encoding the human antibody lightchain, and a gene encoding the non-human animal-derived IgM constantregion, and also comprising a gene encoding the human antibody surrogatelight chain of the present invention may be constructed by using themethods of above (1) to (4).

Specifically, the vector of the present invention may be constructed bysubstituting the human IgM heavy chain constant region gene on the humanchromosome 14 fragment comprising the human antibody heavy chain geneconstructed by the method of (1) with the non-human animal-derived IgMheavy chain constant region gene by the method of (3); and then ligatingthe human chromosome 2 fragment comprising the human κ chain geneconstructed by the method of (2), and the human chromosome 22 fragmentcomprising the human λ chain gene and the human antibody surrogate lightchain gene constructed by the methods of (2) and (4).

More specifically, a human artificial chromosome vector comprising thehuman antibody heavy chain gene, the human antibody light chain gene,and the human antibody surrogate light chain gene of the presentinvention, and also comprising the non-human animal-derived 1gM constantregion may be constructed in the following manner.

First, the DNAs encoding the DNAs encoding the CH2 domain, the CH3domain, the CH4 domain, the TM1 domain, and the TM2 domain in the humanIgM heavy chain constant region gene on the human chromosome 14 fragmentare substituted with the DNAs encoding the CH2 domain, the CH3 domain,the CH4 domain, the TM1 domain, and the TM2 domain of the non-human IgMheavy chain constant region gene, respectively, by homologousrecombination. Subsequently, a loxP sequence is inserted into the RNR2gene locus (Worton et al., Science, 239, 64-68, 1988) on the humanchromosome 14 fragment by homologous recombination.

Meanwhile, a loxP sequence and a lox2272 sequence which are recognitionsequences of a Cre recombinant enzyme, are inserted into the cos138 site(Kuroiwa, Nature Biotechnology, 27, 173-181, 2009) which is positionedon the polar centromere side and into the AC104134 site (Gene AccessionNo.) which is positioned on the polar telomere side of the human K chaingene cluster region on the human chromosome 2 fragment, respectively, byhomologous recombination.

Further, after a telomere sequence is inserted into the AP000350 site(Gene Accession No.) on the polar telomere side of a cluster regioncomprising the human λ chain gene and the human antibody surrogate lightchain gene on the human chromosome 22 fragment by homologousrecombination and then cut the chromosome, a lox 2272 sequence isinserted into the AP000553 site (Gene Accession No.) on the polarcentromere side by homologous recombination.

By Cre/loxP recombination, both chromosomes are ligated by translocatingthe AP000553 site on the human chromosome 22 fragment into the AC104134site on the human chromosome 2 fragment. In addition, by Cre/loxPrecombination, three chromosomes are ligated by translocating the RNR2gene locus on the human chromosome 14 fragment into the cos138 site onthe human chromosome 2 fragment of the above ligated bodies.

Examples of the human artificial chromosome vector of the presentinvention to be constructed in this manner include cKSL-HACΔ. In FIG. 3,a schematic view of cKSL-HACΔ is shown. In FIG. 3, a schematic view ofcKSL-HACΔ is shown. As shown in FIG. 3, since cKSL-HACΔ furthercomprises a gene encoding the non-human animal (bovine)-derived IgMheavy chain constant region and a gene encoding the human antibodysurrogate light chain compared to the structure of κHAC (FIG. 1), it canbe produce human antibodies with higher efficiency when it is introducedinto an animal, and an animal which is capable of producing humanantibodies with such a high efficiency may also be stably produced.

3. An Animal Having the Human Artificial Chromosome Vector of thePresent Invention

The animal having the human artificial chromosome vector of the presentinvention refers to an animal into which the human artificial chromosomevector of the present invention is introduced.

The animal having the human artificial chromosome of the presentinvention is not particularly limited so long as the animal is an animalin which the human artificial chromosome fragment may be introduced intoa cell thereof, and any non-human animals, for example, ungulates suchas cows, horses, goats, sheep, and pigs; rodents such as mice, rats, andrabbits; poultry such as chickens, domestic ducks, and geese; and thelike may be used.

The non-human animal is preferably a non-human mammalian animal, morepreferably an ungulate animal, and even more preferably a bovine.

An animal having the human artificial chromosome vector of the presentinvention may be constructed by introducing the human artificialchromosome vector of the present invention constructed by the method ofthe above (2) into an oocyte of a host animal.

Specifically, the human artificial chromosome vector of the presentinvention to be constructed by the method of the above (2) using themethod described in WO2005/104835 and the method of Kuroiwa et at.(Kuroiwa et al., Nature Biotechnology, 20, 889-894) is introduced into asomatic cell derived from a host animal by a microcell fusion method.Thereafter, the animal having the human artificial chromosome vector maybe constructed by transplanting a nucleus or chromatin agglomerate ofthe cell into an oocyte and transplanting the oocyte or an embryo to beformed from the oocyte into the uterus of a host animal to give birth.

It may be confirmed by a method of Kuroiwa et al. (Kuroiwa et al.,Nature Biotechnology, 18, 1086-1090, 2000 and Kuroiwa et al., NatureBiotechnology, 20, 889-894) whether an animal constructed by the abovemethod has the human artificial chromosome vector of the presentinvention.

4. Method for Producing a Human Antibody of the Present Invention

A antigen-specific human antibody may be produced by immunizing theanimal having the human artificial chromosome vector of the presentinvention constructed in the above (3) with a desired antigen to producethe antigen-specific human antibody in the serum of the animal andrecovering the antigen-specific human antibody from the serum.

The antigens for immunizing the animal having the human artificialchromosome vector of the present invention, are not particularly limitedand examples include a tumor-associated antigen, an antigen associatedwith allergy or inflammation, an antigen associated with cardiovasculardisease, an antigen associated with autoimmune disease, an antigenassociated with neurodegenerative disease, and an antigen associatedwith viral or bacterial infections.

Examples of tumor-associated antigens include CD1a, CD2, CD3, CD4, CD5,CD6, CD7, CD9, CD10, CD13, CD19, CD20, CD21, CD22, CD25, CD28, CD30,CD32, CD33, CD38, CD40, CD40 ligand (CD40L), CD44, CD45, CD46, CD47,CD52, CD54, CD55, CD55, CD59, CD63, CD64, CD66b, CD69, CD70, CD74, CD80,CD89, CD95, CD98, CD105, CD134, CD137, CD138, CD147, CD158, CD160,CD162, CD164, CD200, CD227, adrenomedullin, angiopoietin related protein4 (ARP4), aurora, B7-H1, B7-DC, integlin, bone marrow stromal antigen 2(BST2), CA125, CA19.9, carbonic anhydrase 9 (CA9), cadherin,cc-chemokine receptor (CCR) 4, CCR7, carcinoembryonic antigen (CEA),cysteine-rich fibroblast growth factor receptor-1 (CFR-1), c-Met, c-Myc,collagen, CTA, connective tissue growth factor (CTGF), CTLA-4,cytokeratin-18, DF3, E-catherin, epidermal growth facter receptor(EGFR), EGFRvIII, EGFR2 (HER2), EGFR3 (HER3), EGFR4 (HER4), endoglin,epithelial cell adhesion molecule (EpCAM), endothelial protein Creceptor (EPCR), ephrin, ephrin receptor (Eph), EphA2, endotheliase-2(ET2), FAM3D, fibroblast activating protein (FAP), Fe receptor homolog 1(FcRH1), ferritin, fibroblast growth factor-8 (FGF-8), FGF8 receptor,basic FGF (bFGF), bFGF receptor, FGF receptor (FGFR)3, FGFR4, FLT1,FLT3, folate receptor, Frizzled homologue 10 (FZD1O), frizzled receptor4 (FZD-4), G250, G-CSF receptor, ganglioside (GD2, GD3, GM2, GM3, andthe like), globo H, gp75, gp88, GPR-9-6, heparanase I, hepatocyte growthfactor (HGF), HGF receptor, HLA antigen (HLA-DR, and the like), HM1.24,human milk fat globule (HMFG), hRS7, heat shock protein 90 (hsp90),idiotype epitope, insulin-like growth factor (IGF), IGF receptor (IGFR),interleukin (IL-6, IL-15, and the like), interleukin receptor (IL-6R,IL-15R, and the like), integrin, immune receptor translocationassociated-4 (IRTA-4), kallikrein 1, KDR, KIR2DL1, KIR2DL2/3, KSl/4,lamp-1, lamp-2, laminin-5, Lewis y, sialyl Lewis x, lymphotoxin-betareceptor (LTBR), LUNX, melanoma-associated chondroitin sulfateproteoglycan (MCSP), mesothelin, MICA, Mullerian inhibiting substancetype II receptor (MISIIR), mucin, neural cell adhesion molecule (NCAM),Necl-5, Notchl, osteopontin, platelet-derived growth factor (PDGF), PDGFreceptor, platelet factor-4 (PF-4), phosphatidylserine, ProstateSpecific Antigen (PSA), prostate stem cell antigen (PSCA), prostatespecific membrane antigen (PSMA), Parathyroid hormone relatedprotein/peptide (PTHrP), receptor activator of NF-kappaB ligand (RANKL),receptor for hyaluronic acid mediated motility (RHAMM), ROBO1, SART3,semaphorin 4B (SEMA4B), secretory leukocyte protease inhibitor (SLPI),SM5-1, sphingosine-1-phosphate, tumor-associated glycoprotein-72(TAG-72), transferrin receptor (TfR), TGF-beta, Thy-1, Tie-1, Tie2receptor, T cell immunoglobulin domain and mucin domain 1 (TIM-I), humantissue factor (hTF), Tn antigen, tumor necrosis factor (TNF),Thomsen-Friedenreich antigen (TF antigen), TNF receptor, tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL), TRAIL receptor (DR4,DRS, and the like), system ASC amino acid transporter 2 (ASCT2), trkC,TROP-2, TWEAK receptor Fn14, type IV collagenase, urokinase receptor,vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR1,VEGFR2, VEGFR3, and the like), vimentin, VLA-4, and the like.

Examples of antigens associated with allergy or flare include IL-6,IL-6R, IL-5, IL-5R, IL-4, IL-4R, TNF, TNF receptor, CCR4, chemokine,chemokine receptor, and the like.

Examples of antigens associated with cardiovascular disease includeGPIIb/IIIa, PDGF, PDGF receptor, blood coagulation factor, IgE, α_(v)β₃,α₄β₇, and the like.

Examples of antigens associated with viral or bacterial infectionsinclude gp120, CD4, CCR5, a verotoxin, an anthrax protective antigen, amethicillin-resistant Staphylococcus aureus (MRSA) antigen, a hepatitistype B virus (HBV) antigen, a cytomegalovirus (CMV) antigen, a Rabiesantigen, a Varicella zoster antigen, and the like.

Other examples thereof include a T cell surface membrane proteinmixtures, a Rh (D) antigen, crotalid venom, digoxin, and the like.

The immunization is carried out by administering the antigen with, forexample, a complete Freund's adjuvant or an appropriate adjuvant such asan aluminum hydroxide gel, and pertussis bacteria vaccine,subcutaneously, intravenously, or intraperitoneally into an animal.

Examples of the form of administering the antigen into an animal havingthe human artificial chromosome vector of the present invention includepeptides, protein, bacteria, viruses, cells, biological tissue pieces,and the like.

When the antigen is a partial peptide, a conjugate is produced with acarrier protein such as bovine serum albumin (BSA), keyhole Limpethemocyanin (KLH) or the like, and is used as the immunogen.

The antigen is administered once to 10 times every 1 to 4 weeks afterthe first administration. After 1 to 14 days from each administration,blood is collected from the animal to measure the antibody value of theserum.

Examples of the method for detecting and measuring the antigen-specifichuman antibody included in the serum include a binding assay by anenzyme-linked immunosorbent assay [Antibodies-A Laboratory Manual, ColdSpring Harbor Laboratory (1988)], a biosensor Biacore, and the like.

Specifically, the binding amount of a human antibody in the serum may bemeasured by incubating the serum comprising the human antibody withantigen expressing cells, and then using an antibody specificallyrecognizing a human antibody.

Further, in addition to these methods, the antibody may be selected byidentifying a target antigen of the antibody according to a method knownin the art (The Prostate, 67, 1163, 2007).

Examples of the method for recovering human antibodies from the seruminclude a method of purifying by adsorbing the human antibody on aprotein A carrier, a protein G carrier, or a carrier on which the humanimmunoglobulin specific antibody is supported.

Further, methods used in purification of proteins, such as gelfiltration, ion exchange chromatography, and ultrafiltration, may becombined.

A human antibody produced by the above method may be a polyclonalantibody or a monoclonal antibody, and preferably a polyclonal antibody.

EXAMPLE Example 1 Construction of a Targeting Vector

(1) Construction of a Targeting Vector pTElL′hisDpuro^(lox2272)F9R9 x

Methods described in the publication (Kuroiwa et al., Nat Biotechnol.18: 1086-1090, 2000, Kuroiwa et al., Nat Biotechnol. 20: 889-894, 2002and Kuroiwa et al., Nat Biotechnol. 27: 173-181, 2009) were basicallyused for construction of a targeting vector.

Specifically, a genomic DNA fragment Dk-F9R9 used as a homology arm wasamplified by PCR consisting of 40 cycles of 98° C. for 10 seconds and68° C. for 9 minutes by using two primer DNAs of kD-F9(5′-tcgaggatccgccagggagacagatgccaagtacggtttag-3′) (SEQ ID NO:1) andkD-R9 (5′-tcgaggatccaggatctttgggggactgaatggggtgtgct-3′) (SEQ ID NO:2)and a genomic DNA of a chicken DT40 cell line KTL1 (Kuroiwa et al., Nat.Biotechnol. 27: 173-181, 2009) having the human chromosome 2 as atemplate.

Subsequently, the plasmid pTEL′hisDpuro^(lox72272) was constructed inthe following order.

First, the plasmid pPUR^(lox2272) was constructed by annealing two oligoDNA fragments [a DNA fragment consisting of a nucleotide sequence of5′-agcttggatccataacttcgtataggatactttatacgaagttata-3′ (SEQ ID NO:3) and aDNA fragment consisting of a nucleotide sequence of5′-agcttataacttcgtataaagtatcctatacgaagttatggatcca-3′ (SEQ ID NO:4)]comprising a modified type lox2272 sequence, followed by cloning intothe HindIII site of the plasmid pPUR (BD Bioscience Clontech).

Meanwhile, the plasmid pTEL′hisDPm was constructed by substituting thepuromycin resistance gene (hereinafter referred to as puro′) of theplasmid pTELpuro (Kuroiwa et al., Nat Biotechnol. 18: 1086-1090, 2000,Kuroiwa et al., Nat Biotechnol. 20: 889-894, 2002, and Kuroiwa et al.,Nat Biotechnol. 27: 173-181, 2009) with the hisD gene, substituting theEcoRI site with the SrfI site, and substituting the SpeI site with thePmeI site.

After generating the BamHI-digested fragment of the

2272 blunt ended, the obtained fragment was cloned into the PmeI site ofthe pTEL′hisDPm and thus obtained plasmid was named as thepTEL′hisDpuro^(lox2272).

The plasmid pTEL′hisDpuro^(lox2272)F9r9 was constructed by subcloningthe Dk-F9R9 amplified by the above PCR into the BamHI site of thepTEL′hisDpuro^(lox2272) (FIG. 4).

(2) Construction of a Targeting Vector pTELCAGzeoSLF2R2

In the same manner as in (1), the pTELCAGzeo(Sr)Pm was constructed bysubstituting the EcoRI site of the plasmid pTELpuro with the SrfI site,then substituting the SrfI site with the PmeI site, and furthersubstituting the puro gene with the CAGzeo gene.

Meanwhile, a genomic DNA fragment used as a homology arm was amplifiedby repeating 40 cycles of 98° C. for 10 seconds and 68° C. for 9 minutesby using SL-F2 (5′-tcgaggatccggcctcccaaaggattatagacgtgagccactgt-3′) (SEQID NO:5) and SL-R2 (5′-tcgaggatccaaagaaggggcccgcctctgcctctaaatcctgac-3′)(SEQ ID NO:6) as a PCR primer set and a chromosome DNA of a chicken DT40cell line 52-18 (Kuroiwa et al., Nucleic Acids Res 26: 3447-3448, 1998)having the human chromosome 22 as a template.

The pTELCAGzeoSLF2R2 was constructed by subcloning the PCR productobtained into the BamHI site of the plasmid pTELCAGzeo(Sr)Pm (FIG. 5).

(3) Construction of a Targeting Vector p553CAG^(lox2272)BsrDT

A vector having a structure in which a targeting vector pHCF2loxPHyg(Kuroiwa et al., Nat Biotechnol. 18: 1086-1090, 2000) was substitutedwith the AP000553 site (GenBank accession Number) sequence in which ahomology arm sequence of the HCF2 gene was amplified by PCR wasconstructed and used as the targeting vector p553loxPHyg(F).

At that time, the amplification of the AP000553 site fragment wascarried out by PCR consisting of 40 cycles of 98° C. for 10 seconds and68° C. for 15 minutes by using 553-F3(5′-tgtagctgactttagccacccacaagtac-3′) (SEQ ID NO:7) and 553-R3(5′-cttgctgattatacctcatctccttccctc-3′) (SEQ ID NO:8) as a primer set anda genomic DNA of the chicken DT40 cell52-18 as a template.

After the obtained plasmid p553loxPHyg(F) was digested with Nod, aself-ligation was carried out, followed by cloning of the diphtheriatoxin A fragment (hereinafter referred to as DT-A) into the Srf site.

Meanwhile, the pDRIVE-CAG (InvivoGen) was modified as follows. The oligoDNAs [5′-gtacaataacttcgtatagcatacattatacgaagttatagatctg-3′ (SEQ ID NO:9)and 5′-aattcagatctataacttcgtataatgtatgctatacgaagttatt-3′ (SEQ ID NO:10)]each comprising the loxP sequence were annealed and the lacZ fragment ofthe pDRIVE-CAG was substituted. The pCAGloxP was constructed by cloningthe fragment digested with SdaI and SwaI into the pBluescriptSK-(Stratagene) digested with PstI and SmaI.

Subsequently, the loxP sequence of the pCAG^(loxP) was substituted witha sequence comprising the lox2272 produced by annealing the two oligoDNAs [5′-gatctataacttcgtataggatactttatacgaagttatg-3′ (SEQ ID NO:11) and5′-ctagcataacttcgtataaagtatcctatacgaagttata-3′ (SEQ ID NO:12)]. Further,the pCAG^(lox2272)bsr was constructed by inserting theblasticidin-resistance gene (bsr gene) into the SpeI site.

Finally, the p553CAG^(lox2272)BsrDT (FIG. 6) was completed by cloningthe NotI-KpnI fragment (CAG-lox2272-polyA-bsr) into the NotI site.

(4) Construction of a Targeting Vector pSC355CAG^(lox511)hisDDT

A genomic DNA used as a homology arm was amplified by PCR consisting of40 cycles of 98° C. for 10 seconds and 68° C. for 15 minutes by usingSC355-F3 (5′-gtacaatcttggatcactacaacctctgcctacca-3′) (SEQ ID NO:13) andSC355-R3 (5′-tgctgtgtctaatcaggtgttgaacccatctacta-3′) (SEQ ID NO:14) as aprimer set and a genomic DNA of the chicken DT40 cell comprising thehuman chromosome 14 as a template.

The KpnI site of the plasmid pBluescript was substituted with the SrfIsite, and the DNA fragment amplified above was subcloned into the SpeIsite. The obtained plasmid was used as the pSC355F3R3.

Subsequently, the loxP sequence of the pCAG^(loxP) was substituted witha sequence comprising lox511 produced by annealing the two oligo DNAs [aDNA fragment consisting of the sequence5′-gatctataacttcgtatagtatacattatacgaagttatg-3′ (SEQ ID NO:15) and a DNAfragment consisting of the nucleotide sequence5′-ctagcataacttcgtataatgtatactatacgaagttata-3′ (SEQ ID NO:16)] and usedas the pCAG^(lox511).

The pCAG^(lox511)hisD was constructed by inserting the hisD gene intothe SpeI site of the pCAG^(lox511). A fragment (CAG-lox511-polyA-hisD)digested with NotI and KpnI was cloned into the EcoRV site of thepSC355F3R3. Finally, the plasmid obtained by subcloning the DT-Afragment into the NotI site was used as the pSC355CAG^(lox511)hisDDT(FIG. 7).

(5) Construction of a Targeting Vector p14CEN(FR)hygpuro^(lox511)DT

A genomic DNA used as a homology arm was amplified by PCR consisting of40 cycles of 98° C. for 10 seconds and 68° C. for 15 minutes by using 14CEN-F (5′-tcgaggatccttcgccaccccaaagatgattacagattac-3′) (SEQ ID NO:17)and 14 CEN-R (5′-tcgaggatcctacactagaagcacaaaccccaccattacacat-3′) (SEQ IDNO:18) as a primer set and a genomic DNA of the chicken DT40 cellcomprising the human chromosome 14 as a template.

The p14CEN(FR) was constructed by subcloning the PCR product into theBamHI site of the pBluescript in which the KpnI site was substitutedwith the PmeI site.

The oligo DNAs [a DNA fragment consisting of a nucleotide sequence of5′-agcttggatccataacttcgtatagtatacattatacgaagttata-3′ (SEQ ID NO:19) anda DNA fragment consisting of a nucleotide sequence of5′-agcttataacttcgtataatgtatactatacgaagttatggatcca-3′(SEQ ID NO:20)]comprising the lox511 sequence were annealed. The plasmid pPUR^(lox511)was constructed by cloning the fragment obtained into the HindIII siteof the plasmid pPUR (BD Bioscience Clontech).

The pHygPuro^(lox511) was constructed by cloning the BamHI-digestedfragment of the pPUR^(lox511) into the BamHI site of the pBluescript,and the hygromycin resistance gene (hyg gene) into the EcoRV site,respectively.

A fragment (puro-lox511-hyg) digested with NotI and KpnI was cloned intothe HpaI site of the p14CEN(FR). Finally, thep14CEN(FR)hygpuro^(lox511)DT (FIG. 8) was completed by subcloning theDT-A fragment into the PmeI site.

(6) Construction of a Targeting Vector pRNR2^(loxP)bsrDT

The targeting vector pRNR2^(loxP)bsrDT (FIG. 9) was constructed byinserting the DT-A fragment into the vector pRNR2^(loxP)bsr (Kuroiwa etal., Nat Biotechnol. 18: 1086-1090, 2000).

(7) Construction of a Targeting Vector pCH1CAGzeoDT

A λ phage genomic library of κHAC (WO2009/111086) was constructed fromthe CHO cell comprising κHAC by a Custom Library Construction Service(Loftstrand Labs, Ltd.) by using the λFIX II vector.

A clone comprising the human IgM constant region from the constructedgenomic library was screened by using, as a probe, a PCR productamplified by PCR consisting of 40 cycles of 98° C. for 10 seconds, 64°C. for 30 seconds, and 72° C. for 1 minute with DNAs consisting ofnucleotide sequences of 5′-cagtccccggcagattcaggtgtcc-3′ (SEQ ID NO:21)and of 5′-gaaagtggcattggggtggctctcg-3′ (SEQ ID NO:22) as a primer and achromosome DNA extracted from the CHO cell (WO2009/111086) comprisingκHAC as a template. As a result, clones #1, #4, and #7 were isolated.

Clone #4 (the PmlI fragment, 1.7 kb) was subcloned into the SmaI site ofthe pBluescript and was named as pCH1S(F). pCH1SSP(F) was constructed bysubcloning the SacI-PmlI fragment (1 kb) derived from the plasmidpBCμAY37-95 in which the SacI-bovine IGHM chromosome fragment was clonedinto the pBluescript, into the PstI site of pCH1S(F).

pCH1SL was constructed by cloning the SmaI-EcoRI fragment (7.4 kb)derived from clone #1 into pCH1SSP(F) digested with EcoRV/EcoRI.

Meanwhile, from the pBCμAY37-95, the SacI-digested fragment (3.5 kb) waspCH2S(F) was subcloned into the pBluescript. pmAYSazeo(F) wasconstructed by cloning the XhoI-digested fragment (the fragmentconstructed by inserting the CAGzeo fragment into the EcoRV site ofpBS246 (Gibco) and digesting with XhoI) of CAGzeo into which the loxPsequence was introduced, into the Van911 site of the obtained plasmid.

Further, pCH1zeo(F) was constructed by subcloning the SacI fragment ofprnAYSazeo(F) into the blunt-ended EcoRI site of pCH1SL. Finally, thepCH1CAGzeoDT (FIG. 10) was completed by subcloning the DT-A fragmentinto the NotI site of pCH1zeo(F).

(8) Construction of a Targeting Vector pCH2CAGzeoDT

The Se Sp fragment produced by annealing the oligo DNAs consisting ofnucleotide sequences of5′-ggaccaggtggagactgtgcagtcctcacccataactttcagggcctacagcatgctg-3′ (SEQ IDNO:23) and5′-cagcatgctgtaggccctgaaagttatgggtgaggactgcacagtctccacctggtcc-3′ (SEQ IDNO:24) was cloned into the blunt-ended PstI site of pBluescript.

pmAYSpB was constructed by subcloning a fragment (about 2 kb) digestedwith SphI and BamHI from the plasmid pBCμAY37-95 into the SphI-BamHIsite of the plasmid obtained above.

Similarly, the pmAYSpBPml was constructed by subcloning a fragment(about 2 kb) digested with BamHI and PmlI of the pBCμAY37-95 into theBamHI-PmlI site (with which the original SpeI site is substituted) ofthe prnAYSpB.

The pRISe was constructed by subcloning the EcoRI-SexAI fragment (about0.6 kb) of clone #1 in Example I (7) into the EcoRI-SexAI site of thepmAYSpBPml.

Subsequently, the pRISeCAGzeo(R) was constructed by subcloning theCAGzeo into which the loxP sequence was introduced, into the Van911 siteof the pRISe. Further, the pRISeCAGzeoE was constructed by substitutingthe NotI site of the pRISeCAGzeo(R) with the EcoRI site.

Meanwhile, the pCH2S(F) was constructed by subcloning the PmlI fragment(about 1.7 kb) of clone #4 in Example I(7) into the SmaI site of thepBluescript in which the EcoRV site was substituted with the MluI site.

The pCH2LS was constructed by cloning a fragment (about 6.6 kb) digestedwith MluI and EcoRI of clone #1 in Example 1(7) into the MluI-EcoRI siteof the pCH2S(F).

Subsequently, the pCH2CAGzeo(F) was constructed by subcloning the EcoRIfragment of the pRISeCAGzeoE into the EcoRI site of the pCH2LS. Finally,the pCH2CAGzeoDT (FIG. 11) was completed by subcloning the DT-A fragmentinto the NotI site of the pCH2CAGzeo(F).

Example 2 Construction of KSL-HAC (1) Modification of the HumanChromosome 2 in a Chicken DT40 Cell

In order to generate a deletion at the AP104134 site of the humanchromosome 2 and insert a lox2272 sequence and a promoterless puro^(r)cassette, the targeting vector pTEL′hisDpurolox2272F9R9 was linearizedwith SrfI (Stratagene) and introduced into KTL1 (Kuroiwa et al., Nat.Biotechnol. 27: 173-181, ×2009) which was a chicken DT40 cell linehaving the human chromosome 2 fragment digested at the CD8A gene locusby electroporation (550 V, 25 μF). The electroporation of the DT40 cellwas carried out by a method described in the publication (Kuroiwa etal., Nat. Biotechnol. 18: 1086-1090, 2000).

Colonies were subjected to selection by histidinol (0.5 mg/ml, Sigma)for two weeks and the sensitivity to puromycin (1 μg/ml, Sigma) wasmeasured as an index of deletion of the puro^(r) cassette on the CD8Agene locus. The chromosome DNA was extracted from a colony having thepuromycin sensitivity by using the Gentra Puregene cell kit (QIAGEN),and was subjected to a PCR screening using the FABP1-F(5′-tatcaagggggtgtcggaaatcgtg-3′) (SEQ ID NO:25) and the FABP1-R(5′-actgggcctgggagaacctgagact-3′) (SEQ ID NO:26) as primers.

PCR was carried out under a condition of amplifying the FABP1 gene locuswhich was present in KTL1 but was not present at a target clone byrepeating 30 cycles of 98° C. for 10 seconds, 60° C. for 30 seconds, and72° C. for 1 minute. As a result, clone K53 was identified as a clone inwhich a desired deletion occurred. FIG. 12 illustrates an outline of amethod for modifying the human chromosome 2 in a chicken DT40 cell.

(2) Modification of the Human Chromosome 22 in a Chicken DT40 Cell

In order to generate a deletion at the AP000350 site which is positionedon about 450 Mb telomere side from the AP000344 site (Kuroiwa et al.,Nat. Biotechnol. 20: 889-894, 2002) of the human chromosome 22, thetargeting vector pTELCAGzeoSLFR was linearized with PmeI (New EnglandBiolabs) and introduced into 52-18 (Kuroiwa et al., Nucleic Acids Res26: 3447-3448, 1998) which is a chicken DT40 cell line having the humanchromosome 22 by electroporation (550 V, 25 μF).

A colony was subjected to selection by Zeocin (1 mg/ml, Invitrogen) for2 weeks. A genomic DNA extracted from the thus obtained colony wassubjected to PCR screening by using 350T-F(5′-gaggtgggctgaggggcaagtgtg-3′) (SEQ ID NO:27) and 350T-R(5′-tacgaggaggggaggcagtgagagg-3′) (SEQ ID NO: 28) as primers.

PCR was carried out under conditions (repeating 30 cycles of 98° C. for10 seconds, 63° C. for 30 seconds, and 72° C. for 1 minute) in which theAP000350 site which was present at 52-18 but was not present at a clonein which the targeting occurred, was amplified. As a result, it wasfound that a digestion was generated exactly in clone ST13.

In order to integrate a lox2272 sequence and a CAG promoter into theAP000553 site, the targeting vector p553CAGlox2272bsrDT which waslinearized with PmeI (New England Biolabs) was introduced into ST13 byelectroporation (550 V, 25 μF).

A colony was subjected to selection by blasticidin S (15 μg/ml,Invitrogen) for 2 weeks. A genomic DNA was extracted from the thusobtained colony and was subjected to PCR screening by using 553KO-F(5′-gtcagccaggcgggccatttaccgtaagttatgta-3′) (SEQ ID NO: 29) and 553KO-R(5′-agggctgggttagatggcaccaaatgaaaggagaa-3′) (SEQ ID NO: 30) as primers.

PCR was carried out by repeating 40 cycles of 98° C. for 10 seconds and68° C. at 6 minutes. As a result, clone STL54 was identified as a clonein which a targeting occurred. FIG. 13 illustrates an outline of amethod for modifying the human chromosome 22 in a chicken DT40 cell.

(3) Construction of a SLKH Fragment in a DT40 Hybrid Cell

A SLKH fragment was constructed in a chicken DT40 hybrid cell accordingto a method of the publication (Kuroiwa et al., Nat. Biotechnol. 27:173-181, 2009).

The K53 (Kuroiwa et al., Nat. Biotechnol. 27: 173-181, 2009) comprisinga fragment derived from the human chromosome 2 having a hyg^(r) cassetteprepared in Example 2(1) and the STL54 comprising a fragment derivedfrom the human chromosome 22 having a bs^(r) cassette prepared inExample 2(2) were fused by using the PEG1500 (Roche) to prepare a DT40hybrid cell.

The colony was maintained in the presence of hygromycin B (1.5 mg/ml,Invitrogen) and blasticidin S (20 μg/ml, Invitrogen) for 3 weeks toselect a cell maintaining both human chromosome fragments. A genomic DNAwas extracted from the colony and was subjected to PCR.

It was confirmed whether the human chromosome 2 fragment was maintainedby carrying out PCR which repeated 40 cycles consisting of 98° C. for 10seconds and 65° C. for 8 minutes when cos138KO-F and cos138KO-R wereused and 40 cycles consisting of 98° C. for 10 seconds, 60° C. for 30seconds, and 72° C. for 1 minute for other cases, respectively, usingprimer combinations shown in Table 1.

TABLE 1 Combinations of Primers of PCR PrimerNucleotide sequence of primer PCR 1 IGKC-F 5′-tggaaggtggataacgccct-3′(SEQ ID NO: 31) IGKC-R 5′-tcattctcctccaacattagca-3′ (SEQ ID NO: 32)PCR 2 IGKV-F 5′-agtcagggcattagcagtgc-3′ (SEQ ID NO: 33) IGKV-R5′-gctgctgatggtgagagtga-3′ (SEQ ID NO: 34) PCR 3 RPIA-F5′-cttacccaggctccaggctctatt-3′ (SEQ ID NO: 35) RPIA-R5′-ctctacctccctaccccatcatcac-3′ (SEQ ID NO: 36) PCR 4 EIF2AK3-F5′-aggtgctgctgggtggtcaagt-3′ (SEQ ID NO: 37) EIF2AK3-R5′-gctcctgcaaatgtctcctgtca-3′ (SEQ ID NO: 38) PCR 5 cos138KO-F5′-tctttctctcacctaattgtcctggc-3′ (SEQ ID NO: 39) cos138KO-R5′-aggactggcactcttgtcgatacc-3′ (SEQ ID NO: 40)

It was confirmed whether the human chromosome 22 fragment was maintainedby carrying out PCR by the following reaction cycle, for each case witha combination of primers shown in the following Table 2.

For each PCR reaction, PCR1 and 7 were carried out at 40 cycles of 98°C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute, PCR2and 3 were carried out at 40 cycles of 98° C. for 10 seconds, 63° C. for30 seconds, and 72° C. for 1 minute, PCR4 and 5 were carried out at 40cycles of 98° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 1minute, PCR6 was carried out at 40 cycles of 98° C. for 10 minutes, 65°C. for 30 seconds, and 72° C. for 1 minute, and PCR8 was carried out at40 cycles of 98° C. for 10 seconds and 68° C. for 6 minutes.

TABLE 2 Combinations of Primers for PCR PrimerNucleotide sequence of primer PCR 1 553P-F5′-agatctcttgagcccagcagtttga-3′ (SEQ ID NO: 41) 553P-R5′-tgaagttagccggggatacagacg-3′ (SEQ ID NO: 42) PCR 2 hVpreB1-F5′-tgtcctgggctcctgtcctgctcat-3′ (SEQ ID NO: 43) hVpreB1-R5′-ggcggcggctccaccctctt-3′ (SEQ ID NO: 44) PCR 3 hVpreB3-F5′-cactgcctgcccgctgctggta-3′ (SEQ ID NO: 45) hVpreB3-R5′-gggcggggaagtgggggagag-3′ (SEQ ID NO: 46) PCR 4 IgL-F5′-ggagaccaccaaaccctccaaa-3′ (SEQ ID NO: 47) IgL-R5′-gagagttgcagaaggggtgact-3′ (SEQ ID NO: 48) PCR 5 344-F5′-atcatctgctcgctctctcc-3′ (SEQ ID NO: 49) 344-R5′-cacatctgtagtggctgtgg-3′ (SEQ ID NO: 50) PCR 6 hL5-F5′-agccccaagaacccagccgatgtga-3′ (SEQ ID NO: 51) hL5-R5′-ggcagagggagtgtggggtgttgtg-3′ (SEQ ID NO: 52) PCR 7 350P-F5′-accagcgcgtcatcatcaag-3′ (SEQ ID NO: 53) 350P-R5′-atcgccagcctcaccatttc-3′ (SEQ ID NO: 54) PCR 8 553KO-F5′-gtcagccaggcgggccatttaccgtaag ttatgta-3′ (SEQ ID NO: 55) 553KO-R51-agggctgggttagatggcaccaaatgaa aggagaa-3′ (SEQ ID NO: 56)

Further, a fluorescence in situ hybridization (FISH) using the HumanCot-1 DNA (Invitrogen) as a probe was carried out according to a methodin the publication (Kuroiwa et al., Nat. Biotechnol. 18: 1086-1090,2000), and it was confirmed that two human chromosome fragments hadappropriate sizes (the human chromosome 2 fragment was about 154 Mb andthe human chromosome 22 fragment was about 24 Mb of). As a result, cloneSLK2 was identified as a positive clone.

In order to cause a site specific recombination among two lox2272 sites,the AC104134 site on the human chromosome 2 fragment and the AP000553site on the human chromosome 22 fragment, the Cre expression plasmid wasintroduced into SLK2 by electroporation (550 V, 25 JμF).

A recombinant was selected in the presence of puromycin (1 to 5 μg/ml,Invitrogen) for 10 days by using the puromycin resistance imparted bythe CAG promoter lox2272-puro^(r) cassette formed at the translocationposition.

Further, it was confirmed that the recombination was occurred by PCR (40cycles of 98° C. for 10 seconds and 68° C. for 1.5 minutes) usingCAGpuro-F3 (5′-gcggcgccggcaggaaggaaatg-3′) (SEQ ID NO:57) and CAGpuro-R3(5′-cgaggcgcaccgtgggcttgta-3′) (SEQ ID NO:58) as primers and sequencinganalysis of PCR products.

As a result, SLKH6 was identified as a clone in which a desiredtranslocation was occurred to maintain the SLKH fragment. FIG. 14illustrates an outline of a method for constructing the SLKH fragment ina DT40 hybrid cell.

Example 3 Construction of CH2D (1) Modification 1 of the HumanChromosome 14 in a DT40 Cell

In order to integrate a lox511 sequence and a CAG promoter on theAL512355 site which is positioned on the centromere side about 300 kbfrom the IgH gene locus, the targeting vector pSC355CAGlox511hisDDTlinearized with SrfI (Stratagene) was introduced into the DT40 cellmaintaining an intact human chromosome 14 labeled with 35 pSTneo [Katohet al., Cell Structure and Function, 12, 575-580, 1987; JapaneseCollection of Research Biologicals(JCRB) Bank, Deposit Number VE039] byelectroporation (550 V, 25 μF). The electroporation method into the DT40cell is described in the publication (Kuroiwa et al., Nat. Biotechnol.18: 1086-1090, 2000).

The colony was subjected to selection by histidinol (0.5 mg/ml, Sigma)for two weeks, and thus a genomic DNA extracted from the resistantcolony was subjected to PCR screening.

PCR was carried out with 40 cycles of 98° C. for 10 seconds and 68° C.for 6 minutes by using primers amplifying a clone in which the targetingoccurred SC355KO-F2 (5′-acggcgtgaggaccaaggagcgaaacc-3′) (SEQ ID NO:59)and SC355KO-R2 (5′-tgagcgacgaattaaaacaggcgatgac-3′) (SEQ ID NO:60) andalso with 40 cycles of 98° C. for 10 seconds, 60° C. for 30 seconds, and72° C. for 1 minute by using primers amplifying a clone in which vectorfragments were randomly integrated 355N-F(5′-gggcaacatagcaagacaccattc-3′) (SEQ ID NO:61) and 355N-R(5′-tcctctcacctcagcctccatagta-3′) (SEQ ID NO:62).

As a result, clone 1355-2 was identified as a clone which generated thetargeting.

Subsequently, in order to insert a lox511 sequence and a promoter lesspuro^(r) cassette into the AL391156 region on the centromere side 85 Mbfrom the AL512355 site, the targeting vector p14CEN(FR)hygpuro lox511DTlinearized with NotI (Roche) was introduced into 1355-2 byelectroporation (550 V, 25 μF).

The colony was subjected to selection by hygromycin (1.5 mg/ml,Introgen) for two weeks, and thus a genomic DNA extracted from theresistant colony was subjected to PCR screening. It was determined byPCR under conditions of 40 cycles of 98° C. for 10 seconds and 68° C.for 5 minutes by using the primers 14CENKO-F3(5′-actgaaatattttaaatgtttgcccttcccactcc-3′) (SEQ ID NO:63) and14CENKO-R3 (5′-agacctccgcgccccgcaacctccccttctac-3′) (SEQ ID NO:64) onesin which the targeting occurred.

Further, a random insertion was determined by PCR under conditions of 30cycles of 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 1minute by using the primers 14CEN(N)-F2 (5′-aacagttgaatttatggggagtc-3′)(SEQ ID NO:65) and 14CEN(N)-R2 (5′-tcaggctttaaacacagtatcacag-3′) (SEQ IDNO:66).

As a result, clone I156-10 was identified as a clone in which thetargeting occurred.

In order to achieve a reduction from about 106 Mb to 21 Mb by deleting asequence of about 85 Mb from the human chromosome 14 by generating asite specific recombination between two lox511 sites each disposed onthe AL512355 site and the AL391156 site, a Cre expression plasmid wasintroduced into the I156-10 by electroporation (550 V, 25 μF).

Since the puromycin resistance was imparted by a CAGpromoter-lox511-puro^(r) cassette formed at a recombination site, thecolony was cultured for 4 days and was subjected to selection bypuromycin (5 μg/ml, Sigma). The sequences of PCR products were analyzedby carrying out PCR using the primers CAGpuro-F3 and CAGpuro-R3described in Example 2(3), and thus the presence of the cassette wasconfirmed.

Further, in order to confirm that both of hisD and hyg^(r) cassettes waslost as a result of deletion of the 85 Mb, the sensitivity to histidinol(0.5 mg/ml, Sigma) and hygromycin (1.5 mg/ml, Invitrogen) was analyzed.In addition, it was confirmed that the human chromosome 14 was shortenedby FISH using the Human Cot-1 DNA (Invitrogen) as a probe.

In the above, clone D8 was identified as a clone in which a desireddeletion was achieved.

In order to integrate a loxP sequence into a GFP code sequence on theRNR2 gene locus (Kuroiwa et al., Nat. Biotechnol. 27: 173-181, 2009) ofthe human chromosome 14, the targeting vector pRNR2loxPbsrDT linearizedwith SwaI (Roche) was introduced into clone D8 by electroporation (550V, 25 μF). The colony was subjected to selection by blasticidin S (20μg/ml, Invitrogen) for two weeks. A genomic DNA of the resistant colonywas subjected to PCR screening using the primers RNR2-1(5′-tggatgtatcctgtcaagagacc-3′) (SEQ ID NO:87) and STOP-3(5′-cagacactctatgcctgtgtgg-3′) (SEQ ID NO:88) with 40 cycles of 98° C.for 10 seconds and 65° C. for 5 minutes.

As a result, clones 14D1 and 14D3 were identified as positive clonesmaintaining the 14D fragment.

In order to construct a chimeric IgM by substituting the CH2 domain tothe TM2 domain of the human immunoglobulin J1 heavy chain constantregion with bovine-derived sequences, the targeting vectorpCH2CAGzeoDT(F) linearized with Sail (Roche) was introduced into clone14D1 by electroporation (550 V, 25 μF).

The colony was subjected to the Zeocin selection of 1 mg/ml for twoweeks, and thus a genomic DNA of the resistant colony was subjected toPCR screening. PCR was carried out under conditions of 40 cycles of 98°C. for 10 seconds and 68° C. for 5 minutes by using the primers cHAC-F(Y-acgcctgctcgcctgcccgctcgcttct-3′) (SEQ ID NO:67) and cHAC-R(5′-ttgccagggccacagttaacggatacg-3′) (SEQ ID NO:68).

The ligated part at 5′ and 3′ of the bovine sequence and the humansequence was confirmed by analyzing the sequences of the PCR products bycarrying out PCR under conditions of 40 cycles of 98° C. for 10 seconds,64° C. for 30 seconds, and 72° C. for 1 minute by using the primers CH25′-F (5′-cagcaccccaacggcaacaaagaaa-3′) (SEQ ID NO:69) and CH2 5′-R(5′-ccccagggctgcactcaccaacat-3′) (SEQ ID NO:70) and of 40 cycles of 98°C. for 10 seconds and 68° C. for 8 minutes by using cHAC-F3(5′-tgcaggtgaagtgacggccagccaagaaca-3′) (SEQ ID NO:71) and cHAC-R3(5′-tggcagcagggtgacagggaaggcagggaaaag-3′) (SEQ ID NO:72) as primers.

In the above, clone CH2D2-4 was identified as a positive clone whichmaintained the CH2D fragment. FIGS. 15 and 16 illustrate an outline of amethod for modifying the human chromosome 14 in a DT 40 cell.

Example 4 Construction of cKSL-HACΔ

(1) Construction of cKSL-HACΔ a DT40 Hybrid Cell

cKSL-HACΔ was constructed in a DT40 hybrid cell according to a methoddescribed in the publication (Kuroiwa et al., Nat Biotechnol. 27:173-181, 2009).

SLKH6 comprising the hyg^(r) cassette in Example 2(3) and CH2D-4comprising the bs^(r) cassette in Example 3(1) were fused by usingPEG1500 (Roche) to construct a DT40 hybrid cell.

The colony was maintained in the presence of hygromycin B (1.5 mg/ml,Invitrogen) and blasticidin S (20 μg/ml, Invitrogen) for 3 weeks toselect a cell maintaining both of the SLKH fragment and the CH2Dfragment. A genomic DNA was extracted from the resistant colony and wassubjected to PCR.

It was confirmed by PCR using the following primers whether the SLKHfragment was maintained. That is, a combination of IGKC-F and IGKC-R, acombination of IGKV-F and IGKV-R, a combination of RPIA-F and RPIA-R, acombination of EIF2AK3-F and EIF2AK3-R, a combination of cos138KO-F andcos138KO-R, a combination of CAGpuro-F3 and CAGpuro-R3, a combination of553P-F and 553P-R, a combination of hVpreBI-F and hVpreBl-R, acombination of hVpreB3-F and hVpreB3-R, a combination of IgL-F andIgL-R, a combination of 344-F and 344-R, a combination of hL5-F andhL5-R, a combination of 350P-F and 350P-R, and a combination of 553KO-Fand 553 KO (all described in Example 2) were used.

It was confirmed by PCR using the following primers whether the CH2Dfragment was maintained. That is, a combination of CAGpuro-F3 andCAGpuro-R3 [described in Example 2(3)], a combination of RNR2-1 andSTOP-3 [described in Example 3(1)], a combination of VH3-F(5′-agtgagataagcagtggatg-3′) (SEQ ID NO:73) and VH3-R(5′-cttgtgctactcccatcact-3′) (SEQ ID NO:74), a combination of g1(g2)-F(5′-accccaaaggccaaactctccactc-3′) (SEQ ID NO:75) and g1(g2)-R(5′-cacttgtactccttgccattcagc-3′) (SEQ ID NO:76), a combination of14CENKO-F3 (5′-actgaaatattttaaatgtttgcccttcccactcc-3′) (SEQ ID NO:89)and 14CENKO-R3 (5′-agacctccgcgccccgcaacctccccttctac-3′) (SEQ ID NO:90),a combination of CH25′-F (5′-cagcaccccaacggcaacaaagaaa-3′) (SEQ IDNO:91) and CH25′-R (5′-ccccagggctgcactcaccaacat-3′) (SEQ ID NO:92), acombination of cHAC-F3 (5′-tgcaggtgaagtgacggccagccaagaaca-3′) (SEQ IDNO:93) and cHAC-R3 (5′-tggcagcagggtgacagggaaggcagggaaaag-3′) (SEQ IDNO:94), and a combination of SC355F3R3KO-F2 (5′-gccattgtcgagcaggtagt-3′)(SEQ ID NO:95) and SC355F3R3KO-R2 (5′-tccctcatcagccatcctaa-3′) (SEQ IDNO:96) were used.

With regard to the PCR conditions, each PCR reaction was carried out at40 cycles of 98° C. for 10 seconds and 68° C. for 1.5 minutes with acombination of CAGpuro-F3 and CAGpuro-R3; at 40 cycles of 98° C. for 10seconds and 65° C. for 5 minutes with a combination of RNR2-1 andSTOP-3; at 40 cycles of 98° C. for 10 seconds, 56° C. for 30 seconds,and 72° C. for 1 minute with a combination of VH3-F and VH3-R; at 40cycles of 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 1minute with a combination of gI(g2)-F and gI(g2)-R; at 40 cycles of 98°C. for 10 seconds and 68° C. for 5 minutes with a combination of14CENKO-F3 and 14CENKO-R3; at 40 cycles of 98° C. for 10 seconds, 64° C.for 30 seconds, and 72° C. for 1 minute with a combination of CH25′-Fand CH25′-R; at 40 cycles of 98° C. for 10 seconds and 68° C. for 8minutes with a combination of cHAC-F3 and cHAC-R3; at 40 cycles of 98°C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for I minute with acombination of SC355F3R3KO-F2 and SC355F3R3KO-R2.

Further, it was confirmed by FISH using the Human Cot-I DNA (Invitrogen)as a probe that two human chromosome fragments had an appropriate size(the SLKH fragment was about 156 Mb and the CH2D fragment was about 21Mb).

In the above, clones cKSLD2 and cKSLD22 were identified as positiveclones.

In order to induce a site specific recombination between two loxP sitesarranged on the cos138 site of the SLKH fragment [Example 2(3)],respectively, and the RNR2 gene locus of the CH2D fragment [Example3(1)]to delete the CAG promoter-zed cassette [Example 3(1)] inserted betweenthe loxP sequences on the chimeric Igμ gene locus, a Cre expressionvector was introduced into cKSLD2 and cKSLD22 by electroporation (550 V,25 μF).

Since the GFP expression capability was imparted by the PGKpromoter-loxP-GFP cassette constructed at the translocation site,recombinants were concentrated by sorting GFP positive cells usingFACSAria according to a method described in the publication (Kuroiwa etal., Nat. Biotechnol. 18: 1086-1090, 2000).

Sorting was carried out twice to obtain a GFP positive cell group havingtwo different GFP expression levels.

It was confirmed by PCR of 40 cycles of 98° C. for 10 seconds and 68° C.for 4 minutes by using the PCR primers PGK2(5′-tgttctcctcttcctcatctcc-3′) (SEQ ID NO:79) and GFP2(5′-tgaaggtagtgaccagtgttgg-3′) (SEQ ID NO:80) and the PCR primersCreCAGzeo-F3 (5′-gccctcaccttgcagaccacctccatcat-3′) (SEQ ID NO:77) andCreCAGZeo-R3 (5′-cctctcctgctcagtccccttccttccatc3′) (SEQ ID NO:78) thatthe CAG promoter-zeo^(r) cassette on the chimeric lgμ gene locus wasdeleted, and thus it was suggested that a group having a low GFPexpression level maintained cKSL-HACΔ which occurred a desiredtranslocation.

It was confirmed that cKSLD2-derived cKSLDH2(2L) and cKSLD22-derivedcKSLDH22(2L) were identified as DT40 hybrid cell lines consisting ofcell groups which maintained cKSL-HACΔ and exhibited a low GFPexpression level. FIG. 17 illustrates an outline of a method forconstructing cKSL-HACΔ in a DT40 hybrid cell.

(2) Transfer of cKSL-HACΔ from a DT40 Hybrid Cell to a CHO Cell by MMCTMethod

In the following manner, cKSL-HACΔ described in Example 4(1) wastransferred from the DT40 hybrid cell line cKSLDH2(2L) or cKSLDH22(2L)to the Chainese Hamster Ovary (CHO) cell by the MMCT method.

Purified micronuclei derived from cKSLDH2(2L) or cKSLDH22(2L) were fusedwith 2×107 of CHO cells by using PEG1500 (Roche), and a selection wascarried out by Zeocin (800 μg/ml, Invitrogen) and Ouabain (10⁻⁵ M,Sigma) for 3 weeks.

After Zeocin resistant colonies were harvested, a genomic DNA wasextracted, and was subjected to PCR screening. It was confirmed by usingthe following PCR primers whether cKSL-HACΔ was maintained. That is, acombination of IGKC-F and IGKC-R, a combination of IGKV-F and IGKV-R, acombination of RPIA-F and RPIA-R, a combination of EIF2AK3-F andEIF2AK3-R, a combination of cos138KO-F and cos138KO-R, a combination ofCAGpuro-F3 and CAGpuro-R3, a combination of 553P-F and 553P-R, acombination of hVpreB1-F and hVpreBl-R, a combination of hVpreB3-F andhVpreB3-R, a combination of IgL-F and IgL-R, a combination of 344-F and344-R, a combination of hL5-F and hL5-R, a combination of 350P-F and350P-R, and a combination of 553KO-F and 553 KO-R [all described inExample 2(3)], a combination of VH3-F and VH3-R, a combination ofg1(g2)-F and g1(g2)-R, and a combination of PGK2 and GFP2, a combinationof 14CENKO-F3 and 14CENKO-R3, a combination of CH25′-F and CH25′-R, anda combination of cHAC-F3 and cHAC-R3 [all described in Example 3(1)], acombination of SC355F3R3KO-F2 and SC355F3R3KO-R2 [Example 3(2)], and acombination of CreCAGzeo-F3 and CreCAGzeo-R3 [all described in Example4(1)] were used.

Further, it was confirmed by FISH using the Human Cot-1 DNA (Invitrogen)as a probe whether cKSL-HACΔ was maintained. In addition, the presenceof the gene loci of the human immunoglobulin heavy chain (IgH), theimmunoglobulin κ chain (Igκ), and the immunoglobulin chain (Igλ) oncKSL-HAC was confirmed by a three-color FISH using the BAC cloneRP11-417P24, RP11-316G9, and RP11-22M5 from Roswell Park CancerInstitute Human Male BAC Library (RPCI-11), (Advanced Geno Techs Co.) asprobes.

A Comparative Genomic Hybridization (CGH) analysis was carried out withRoche NimbleGen by the custom-made array using about 72000 probescovering the gene loci of human IgH, Igκ, and Igλ. As a result, it wasrevealed that clone cKSLDC6 obtained from cKSLD2(2L) by the MMCT methodand clones cKSLDC15 and cKSLDC23 obtained from cKSLDH22(2L) by the MMCTmethod had a homologous structure.

Example 5 Construction of KcHAC (1) Construction of KcHAC in BovineFibroblasts

The bovine fibroblast cell line C537(κHAC^(−/−) IGHM^(−/−) IGHML1^(−/−))was transfected according to a method described in the publication(Kuroiwa et al., Nature Genetics, 36, 775-780, 2004) except that thetargeting vector pCH1CAGzeoDT was used for the Zeocin selection of 400μg/ml.

A recombinant was identified by genome PCR at 40 cycles of 98° C. for 10seconds and 68° C. for 8 minutes using cHAC-F3R3(5′-tgcaggtgaagtgacggccagccaagaaca-3′) (SEQ ID NO:81) and5′-tggcagcagggtgacagggaaggcagggaaaag-3′ (SEQ ID NO:82) as a primer set.

Colony #30 was used for cloning to establish the 40 days gestation fetalmaintaining KcHAC and the cell line C815. FIG. 18 illustrates an outlineof a method for constructing cHAC in fibroblast cells.

(2) Deletion of a CAGzeo Cassette

In order to delete a CAGzeo cassette from KcHAC, the Cre expressionplasmid and pBShisD/Xmni (linear) were introduced together into the cellline C815.

A colony was subjected to selection by hisD (1 mg/ml) for 2 weeks. Aclone in which the CAGzeo cassette was deleted was identified byconfirming that the CAGzeo cassette and the Cre sequence are notcomprised by genome PCR at 40 cycles of 98° C. for 10 seconds, 65° C. or58° C. for 30 seconds, and 72° C. for 30 seconds using zeo-F and zeo-R[consisting of nucleotide sequences of 5′-acgtcgccggagcggtcgagttctg-3′(SEQ ID NO:83) and 5′-tcggccacgaagtgcacgcagttgc-3′ (SEQ ID NO:84),respectively] and Cre-F and Cre-R [consisting of nucleotide sequences of5′-aaaacaggctctagcgttcg-3′ (SEQ ID NO:85) and 5′-ttcggatcatcagctacacc-3′(SEQ ID NO:86), respectively] as primer sets.

Colony #15 was used for cloning to produce the 40 days gestation fetalmaintaining KcHAC in which CAGzeo was deleted and the cell line M112.

(3) Transfer of KcHAC from a Bovine Fibroblast to a CHO Cell by WCFMethod.

A cell line M112 was subjected to WCF with a CHO-K1 cell. Using PEG1500(Roche), 2×10⁶ of each cells were fused and drug selection was carriedout in the presence of G418 (600 μg/ml, Invitrogen) and Ouabain (1×10⁻⁵mol/L, Sigma) for 2 to 3 weeks.

CHO-like cell colonies were subjected to screening by a set of genomePCR using a combination of CH3-F3 and CH4-R2, a combination of VH3-F3R3,a combination of IGKV-FR and IGKC-FR, and a combination of PGK2 and GFP2as primer sets.

Clone CKF4 was identified as a donor for transferring KcHAC to a bovinefibroblast.

Example 6 Transfer of the HAC into a Bovine Cell Line for Generation ofa HAC Bovine for Production of Human IgG

cKSL-HACΔ was transferred from hybrid cells to CHO cells by using theMMCT method (Kuroiwa et al., Nat Biotechnol. 18: 1086-1090, 2000).

The CHO clone comprising the KcHAC was cultured in F12 medium(Invitrogen) supplemented with 10% FBS (Invitrogen) and G418 (0.6 mg/ml)under conditions of 37° C. and 5% C0₂. In addition, the CHO clonecomprising the cKSL-HACΔ was cultured in F12 medium (Invitrogen)supplemented with 10% FBS (Gibco) and Zeocin (0.8 mg/ml) underconditions of 37° C. and 5% CO₂.

A HAC-comprising clone was expanded in culture in 12 of T25 flasks.After the cell density reached 80 to 90%, colcemid (Sigma) was added tothe medium to give a final concentration of 0.1 μg/ml.

After 3 days, the medium was exchanged with a DMEM medium (Invitrogen)supplemented with 10 μg/ml of cytochalasin B (Sigma). Micronuclei wererecovered by centrifuging the flask at 8000 rpm for 60 minutes. Themicronuclei were purified through 8-, 5-, and 3-μm filters (Costar),followed by resuspension in a DMEM medium. The micronuclei were used forfusion with bovine fibroblasts as described below.

Bovine fetal fibroblasts (IGHM^(−/−) IGHMLI^(−/−), Kuroiwa et al., NatBiotechnol. 27: 173-181, 2009) were cultured in α-MEM (Invitrogen)medium supplemented with 15% FBS (Hyclone) under conditions of 38.5° C.and 6.5% CO₂. The fibroblasts were expanded in culture in a T175 flask.When the cell density reached 70 to 80%, the cells were separated fromcells with 0.05% trypsin. The separated fibroblast cells were washedtwice with a DMEM medium and then overlayed on the micronucleisuspension.

After the micronuclei-fibroblast suspension was centrifuged at 1,500 rpmfor 5 minutes, PEG1500 (Roche) was added to the pellet according to theattached protocol to allow the micronuclei to be fused with the bovinefibroblasts.

Subsequently, the fused cells were plated into ten 24-well plates andcultured in an a-MEM medium supplemented with 15% FBS for 24 hours.After that, the medium was exchanged with a medium containing 0.8 mg/mlof G418 in case of KcHAC and 0.6 mg/ml of Zeocin in case of cKSL-HACΔ.

After being cultured in the presence of antibiotic materials for abouttwo weeks, drug-resistant fused cells were selected.

The selected fused cells comprising various HACs were used as achromatin donor, and thus a bovine comprising various HACs was producedby a chromatin transfer method described in WO2002/051997.

Example 7 Evaluation of the Human IgG Level in HAC Bovine Serum

The human IgG levels in various HAC bovine sera constructed weremeasured by a method described in the publication (Kuroiwa et al., NatBiotechnol. 27: 173-181, 2009). The total amount of the human IgG ineach serum of 6-month-old HAC bovines is shown in the following FIG. 19.Further, the average value of the total amount of the human IgG invarious 6-month-old HAC bovine sera is shown in the following Table 3.

TABLE 3 Total Amount of the Human IgG in Bovine Serum of Various6-Month-Old HAC Bovines (μg/mL) κHAC KcHAC cKSL-HACΔ 433.9 1252 4172

As shown in FIG. 19 and Table 3, the amount of human IgG production inserum bovines having KcHAC or cKSL-HACΔ was significantly increased ascompared to bovines having κHAC.

In addition, according to the publication (Kuroiwa et al., NatBiotechnol. 27: 173-181, 2009), the B cell profile in HAC bovineperipheral blood and lymph node was analyzed by flow cytometry. As aresult, the number of B cells expressing IgM in the KcHAC bovine andcKSL-HACbovine peripheral blood was increased as compared to that in theκHAC bovine peripheral blood. Further, the number of B cells expressingIgM even in the KSL-HAC bovine and the cKSL-HACΔ bovine lymph nodes wasincreased as compared to the number in the κHAC bovine lymph node.

Example 8 Production of the Human IgG to the Anthrax Protective Antigenby the HAC Bovines

In order to evaluate that HAC bovines could initiate antigen specifichumoral responses against a known single antigen due to human IgG,anthrax protective antigens (hereinafter referred to as PA) were usedand reviewed. According to a description in the publication (Kuroiwa etal., Nat Biotechnol. 27: 173-181, 2009), three KcHAC/IGHM^(/)IGHML1^(−/−) bovines (No. 1710, No. 1824, and No. 1834) were immunizedwith PA and was measured the titer of PA specific human IgG. The resultsare shown in Table 4.

TABLE 4 Production amount of the PA specific human IgG in the KcHACbovine (U/mg IgG) Second Third Fourth Fifth Immunization ImmunizationImmunization Immunization No. 1710 62607 No. 1824 26481 No. 1834 1197231007 39342 39745

As a result, KcHAC bovines produced PA specific human IgG of about 12000to 63000 U/mg IgG at a time point of the second immunization, and in No.1834, whenever the immunization was repeated to the fifth immunization,the titer production amount of the PA specific human IgG was increased.

Example 9 Production of Human IgG Specific to T Cell Surface MembraneProtein Mixture by HAC Bovines

In order to evaluate that HAC bovines could initiate antigen specifichumoral responses against an unknown complex antigen due to human IgG,aT cell surface membrane protein mixture (fraction of CEM cell membraneformulation, hereinafter referred to as CEM) was used as an antigen andexamined.

(1) Culture of CEM Cell

By using A RPMI1640 medium (ATCC) comprising 10% bovine fetal serum(Hyclone), the human T cell line CCRF-CEM (ATCC) was allowed toproliferate in a constant humidity and temperature chamber at 37° C. and5% C0₂ until the cells in a flask of 225 cm² was confluent (2×10⁶cells/mL).

Into a 850 cm² roller bottle (Corning) with a bent cap, 500 mL of theRPMI1640 medium containing 10% bovine fetal serum was dispensed and thecells were seeded at a density of 2×10⁵ cells/mL.

The roller bottle was put on a roller bottle culturing apparatus (ThermoScientific) and the cells were cultured for 5 days. In order to recoverthe CEM cells from the roller bottle, the culture was poured into a 2 Lsterilized polypropylene biobottle (Nalgen).

Subsequently, the cells were made into a pellet by centrifugation usinga Sorvall RC12BP at 450×g and 2 to 8° C. for 30 minutes. The cell pelletwas resuspended into sterilized iced PBS, followed by washing operationtwice.

After the final washing, the cell pellet was suspended at a density of2×10⁸ cells/mL in an iced lysis buffer (20 mM Tris chloride, 10 mM NaCl,and 0.1 mM MgCl₂) comprising 1 mM PMSF (Sigma), various proteaseinhibitors [1.6 μM Aprotinin, 40 μM Leupeptin, 2 mM AEBSF, 0.1 mMBestatin, 30 μM E-64, and 20 μM PepstatinA (CalBioChem)], and 25.6 μg/mlDNAseI (Sigma). Then, the cells were immediately freezed in liquidnitrogen. The frozen cells were stored at −80° C. until further use.

(2) Isolation of CEM Cell Membrane by Sucrose Density-GradientCentrifugation

The frozen CEM cells were melted in a cooling water bath. In order tobreak up the cell membranes, the CEM cells were subjected toultrasonication twice to three times in a cooling water bath underconditions of 40 amps and 30 seconds by using an ultra sonic processor(Sonics & Materials).

After the crushed material was injected into the bottom of the lysisbuffer in the sterilized ultra clear centrifuge tube with a pipette byusing a cooled 41% (w/v) sucrose solution, centrifugation was carriedout using an ultracentrifuge of Beckman at 83,000×g (SW 32 Ti rotor) for1 hour at 4° C.

A cloud-like intermediate layer comprising the CEM cell membrane whichwas formed between the sucrose layer and the lysis buffer layer wasrecovered and transferred to a polycarbonate ultracentrifuge tube,followed by dilution using a sterilized iced PBS to have a ratio of 1:3.

The CEM membranes were made into a pellet by ultracentrifugation at80,000×g (70.1 Ti rotor) for 50 minutes at 4° C. After supernatant wascarefully removed with a sterilized Pasteur pipette, the CEM membraneswere once washed by ultracentrifugation at 4° C. for 50 minutes by usingsterilized iced PBS.

Finally, the CEM cell membranes were resuspended in PBS. In order tocrush the membrane pellet, the CEM cell membranes were subjected toultrasonication in a cooling water bath under conditions of 20 amps and30 seconds by using an ultrasonic processor. The CEM membrane crushedmaterial was stored at −80° C. until further use.

(3) Preimmunization of CEM Membrane

Against four KcHAC/IGHM^(−/−) IGHMLI^(−/−) bovine (No. 1863, No. 1865,No. 1868, No. 1735) and two cKSL-HAC/IGHM-1-IGHMLr1-bovine (No. 1922,No. 1923), immunization was carried out with a CEM membrane preparationat 3 mg/run.

The CEM membrane preparation was prepared by Montanide ISA 25 adjuvant(Seppic) which was an oil-in-water type emulsion with a saponin-derivedimmune inducer Quil A (Accurate Chemicals). Bovine was immunized fourtimes at intervals of 4 weeks. The vaccine was intramuscularlyinoculated to the cervical region (2 mL/dose).

For measurement of antibody titer, serum samples were collected prior toimmunization and on days 10 and 14 after immunization. After blood wasallowed to stand still in a serum separation tube to clot, and serum wasseparated by centrifugation. Further, the serum was dispensed at 0.5 to1 mL, and freezed and stored until future assay. The titer of anti-CEMantibody was determined by CEM cell specific human IgG ELISA.

(4) CEM Cell Specific ELISA Assay

Four dilution series were prepared by using a 5% membrane Block/PBS (GEHealthcare) buffer from a serum sample.

In order to prepare a standard curve, seven-step concentration serieswhich was diluted by 2.5 times from 275 times to 67,139 times wereprepared by using a serum with a high titer obtained from a CEM immuneanimal of which a final titer determined in advance as an authenticpreparation and using 5% Membrane Block/PBS.

The inverse number of the final concentration was employed as a unit oftiter and a final titer determined in the case of an authenticpreparation was defined as 55,000 units. With regard to positive controlserum and negative control serum, dilution series were prepared with 5%Membrane Block/PBS and used as an internal standard for confirming theconsistency of the assay.

In order to determine the titer of the CEM specific human IgG, 50 μL ofsamples (authentic preparation serum dilution for correction, positivecontrol serum dilution, negative control serum dilution, and measurementsample serum dilution) were injected in duplicate into a U-shaped bottom96 well-microplate (Costar), and 50 μL of the CEM cells (4×10⁶ cells/mL)was added thereto.

After the plate was allowed to stand still at 4° C. for 60 minutes, theplate was washed with 100 to 200 μL of PBS three times to removenon-bound proteins. After each washing operation, the plates weresubjected to centrifugation at 2850×g for 5 minutes to carefully suck inand remove supernatant from each well.

After three washing operations, 100 μL of HRP-labeled donkey anti-humanIgG antibody (Jackson Immuno Research) diluted with 5% MembraneBlock/PBS buffer by 50,000 times was added to each well to resuspend thecell pellet in a HRP solution. After the plate was allowed to standstill at 4° C. for 30 minutes, the plate was washed with PBS three timessame as above.

Finally, the bound anti-CEM antibody was detected by dividing 100μL/well of the TMB+H₂O₂ matrix mixture solution (KPL) into the plate,and the plate was also allowed to stand still at 25° C. for 15 minutes.After the chromogenic reaction was stopped by 100 μL/well of 10%phosphoric acid, 450 nm was measured by using a microplate reader(Biotek Instruments).

A four-parameter standard curve was prepared from values of seven-stepdilution series, and a value of the serum sample was calculated byintrapolation on the curve with Gen5Secure Software. An average titerwas calculated by carrying out a dilution assay three to four times foreach measurement serum sample.

The result is shown in FIG. 20. Further, the titer of the CEM specifichuman IgG in each HAC bovine sera after the second CEM administration isshown in Table 5. After the second CEM administration, it was shown thatthe CEM specific human IgG was produced at about 7000 U/mg IgG in thecKSL-HACbovine, and at about 1700 to 4700 U/mg IgG in the KcHAC bovineamong the total human IgG.

Production of the CEM specific human IgG (U/mg IgG)

Kinds of HAC Bovine No. CEM specific human IgG Cksl-HACΔ No. 19227056.461 No. 1923 7449.184 No 1863 2880.773 KcHAC No. 1865 1691.688 No.1868 1528.455 No. 1735 4697.248

REFERENCE EXAMPLE Reference Example 1 Establishment of Mouse A9 CellMaintaining Human Chromosome 2, 14, and 22

According to a method described in WO1998/037757, a plasmid pSTneo[Katoh et al., Cell Structure and Function, 12, 575-580, 1987; JapaneseCollection of Research Biologicals (JCRB) Bank, Deposit No. VE039] isintroduced into a human normal fibroblast HFL-1 (RIKEN Japan Cell Bank,Deposit No. RCB0251) to obtain a transformed cell.

After that, cell fusion of the transformed cell with a mouse fibroblastA9 (Oshimura et al., Environmental Health Perspectives, 93, 57-58, 1991;JCRB Cell Bank, Deposit No. JCRB0211) is carried out to construct ahybrid cell.

Subsequently, according to a method described in WO01998/037757,micronuclei are prepared from the hybrid cell to be fused with the mouseA9 cell. Each clone comprising desired human chromosome 2, 14, and 22 isidentified from obtained clones by genome PCR, genomic SouthernAnalysis, fluorescence in situ hybridization (FISH), and the like.

Reference Example 2 Construction of the DT40 Hybrid Cell kTL1 Containinga Human Chromosome 2 Fragment

By a micronucleus fusion method described in WO2008/013067, the humanchromosome 2 is introduced from the A9 cell comprising the humanchromosome 2 obtained in Reference Example 1 into the chicken B cellDT40 (JCRB Cell Bank, Deposit No. JCRB2221).

Subsequently, a telomere sequence is inserted into the CD8A gene locuson the human chromosome 2 by introducing a targeting vector pTELPuroCD8A(Kuroiwa et al., Nature Biotechnology, 18, 1086-1090, 2000) into theDT40 hybrid cell by using a telomere truncation method described inWO2008/013067, and the truncation of the chromosome is induced at theinsertion site.

A DT40 hybrid cell kTL1 comprising a human chromosome 2 which has adeleted region from the CD8A gene locus to the telomere end may beconstructed by the manipulation.

Reference Example 3 Construction of the DT40 Hybrid Cell 52-18Containing the Human Chromosome 22

By a micronucleus fusion method described in WO2008/013067, the humanchromosome 22 is introduced from the A9 cell comprising the humanchromosome 22 obtained in Reference Example 1 into the chicken B cellDT40 (JCRB Cell Bank, Deposit No. JCRB2221). A DT40 hybrid cell52-18comprising the human chromosome 22 may be constructed by themanipulation.

Reference Example 4 Construction of the DT40 Hybrid Cell R56 Containinga Human Chromosome 14 Fragment

According to a description of a report by Kuroiwa et al. (Kuroiwa etal., Nature Biotechnology, 18, 1086-1090, 2000), a loxP sequence isinserted into the RNR2 gene locus on the human chromosome 14 byintroducing a targeting vector pRNR2loxPbsr (Kuroiwa et al., NatureBiotechnology, 18, 1086-1090, 2000) into a DT40 cell (InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology (AIST), Deposit No. FERMBP-7583) comprising thehuman chromosome 14 fragment SC20. The DT40 hybrid cell R56 comprisingSC20 which has the loxP sequence at the RNR2 gene locus may beconstructed by the manipulation.

Reference Example 5 Construction of the DT40 Hybrid Cell #14/DT40Containing the Human Chromosome 14

By a micronucleus fusion method described in WO2008/013067, the humanchromosome 14 is introduced from the A9 cell comprising the humanchromosome 14 obtained in Reference Example 1 into the chicken B cellDT40 (JCRB Cell Bank, Deposit No. JCRB2221). The DT40 hybrid cell#14/DT40 comprising the human chromosome 14 may be constructed by themanipulation.

While the present invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope of the presentinvention. In this connection, this application is based on a U.S.provisional application filed on Nov. 17, 2009 (U.S. provisionalapplication No. 61/261,935), the entire contents there of being therebyincorporated by reference.

INDUSTRIAL APPLICABILITY

The present invention provides a human artificial chromosome vectorcomprising a gene encoding the human antibody heavy chain, a geneencoding the human antibody light chain, and a gene encoding IgM heavychain constant region derived from a non-human animal; and being capableof producing a human antibody with a higher efficiency when the vectoris introduced into an animal. By immunizing the animal produced using ahuman artificial chromosome vector of the present invention with adesired antigen, a large quantity of human polyclonal antibodies can besupplied.

1. A human artificial chromosome vector comprising a gene encoding ahuman antibody heavy chain, a gene encoding a human antibody lightchain, and a gene encoding an IgM heavy chain constant region derivedfrom a non-human animal.
 2. The human artificial chromosome vectoraccording to claim 1, comprising a gene encoding a human antibodysurrogate light chain.
 3. The human artificial chromosome vectoraccording to claim 2, in which a gene encoding a human antibodysurrogate light chain is VpreB gene and λ5 gene.
 4. The human artificialchromosome vector according to claim 1, wherein the gene encoding thenon-human animal-derived IgM heavy chain constant region isbovine-derived IGHM.
 5. An animal having the human artificial chromosomevector according to claim
 1. 6. A bovine having the human artificialchromosome vector according to claim
 4. 7. A method for producing ahuman antibody, comprising: administering a target antigen into theanimal according to claim 5 to produce and accumulate the human antibodyspecific to the antigen in serum of the animal, and recovering the humanantibody specific to the antigen from the serum.
 8. A method forproducing a human antibody, comprising: administering a target antigeninto the bovine according to claim 6 to produce and accumulate the humanantibody specific to the antigen in serum of the bovine, and recoveringthe human antibody specific to the antigen from the serum.